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Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy
J. Mol. Biol. (1990) 213, 899-929
Model for the Structure of Bacteriorhodopsin Based on High-resolution Electron Cryo-microscopy R. H e n d e r s o n , J. M . Baldwin, T. A. C e s k a t
MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH, U.K. F. Zemlin, E. B e c k m a n n
Fritz-Haber-Institut der Max-Planck-Gesellschafl Faradayweg 4-6, D-IO00 Berlin 33, F.R.G. and K. H. D o w n i n g
Cell and Molecular Biology Division Lawrence Berkeley Laboratory Berkeley, CA 94720, U.S.A. (Received 20 December 1989; accepted 31 January 1990) The light-driven proton pump-bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full threedimensional transform to 3"5 A. The map of the structure has a resolution of 3"5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven ~-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the fl-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.
tPresent address: EMBL, Heidelberg, F.R.G.
0022-2836/90/120899-31 $03.00/0
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~) 1990AcademicPressLimited
R : Henderson et al.
900 1. Introduction
Bacteriorhodopsin (bRt) is a membrane protein that functions as a light-driven proton pump in Halobacterium halobium (Oesterhelt & Stoeckenius, 1973). The protein forms a 1 : 1 complex with the retinal chromophore, which gives the protein its characteristic purple colour (Oesterhelt & Stoeckenius, 1971). The polypeptide takes up a conformation consisting of seven transmembrane ahelices (Henderson & Unwin, 1975) with the retinal in the middle (Jubb et al., 1984; Seiff et al. 1985). A concise review of our present understanding of the structure of bR, and the role of many of the amino acids in the proton pumping mechanism has been given by Khorana (1988). Briefly, the structure is known at low resolution from the analyses of three independent crystal structures, which have been averaged (Tsygannik & Baldwin, 1987). The amino acid sequence (Ovchinnikov et al., 1979; Khorana et al., 1979), confirmed from the DNA sequence (Dunn et al., 1981), led to a proposal for the path of the polypeptide (Engelman et al., 1980), which also agrees with more recent data (Khorana, 1988). The sequence can be found later, in Figure 19. The orientation of the cytoplasmic surface in the crystal is known from labelling studies and freeze-fracture (Henderson et al., 1978; Hayward et al., 1978), and the retinal position in projection is known from neutron diffraction (Jubb et al., 1984; Seiff et al., 1985; Heyn et al., 1988). In addition, neutron diffraction from specifically deuterated specimens has led to the unequivocal determination of the positions of several of the helices (Engelman & Zaccai, 1980; Trewhella et al., 1986; Popot et al., 1989), again in agreement with the polypeptide connectivity proposed by Engelman et al. (1980). Khorana and his colleagues have investigated a large number of single amino acid mutants of bR, which they have engineered in an Escherichia coli expression system (Khorana, 1988). Specifically, they have shown (Mogi et al., 1988) that mutations to Asp85, Asp96 and, to a lesser extent, Asp212 have profound effects on proton pumping. They have shown (Mogi et al., 1987} that Tyr57 affects the kinetics of chromophore regeneration from free retinal and opsin, and that Tyrl85 has a smail but clear effect on pumping. Just as important, they have shown that changes in any of the other tyrosine or aspartic acid residues do not affect pumping. The absorption spectrum is perturbed by changes to many more amino acid residues including Trp86, Trp137, Trp182 and Trp189 as well as Arg82, Thr89 and Aspll5. Many of these may form the retinal binding site (Khorana, 1988). Mutants defective in proton pumping have been obtained by mutagenesis of bR in Halobacterium strain GRB (Soppa & Oesterhelt, 1989), including changes of Tyr57, tAbbreviations used: bR, baeteriorhodopsin; hR, halorhodopsin; sR, sensory rhodopsin; r.m.s., root-mean-square.
Asp85, Asp96, Trp138 and Trpl0 (Soppa et al., 1989). Such data provide strong evidence to indicate the orientations of many of the a-helices in the structure, showing which "side-chains must point inwards towards the retinal binding site and the pathway or channel through which the proton must move. In general, orientations derived from mutagenesis are in agreement with the idea that the helices should be oriented with their hydrophobic faces pointing outwards towards the lipid and their hydrophilic faces pointing inwards to the active site (Engelman & Zaccai, 1980; Argos et al., 1982). Two other retinal proteins from halobacteria, together with bR, make up a family of homologous membrane proteins. They are the light-driven chloride ion pump, halorhodopsin (hR) and the phototactic pigment, sensory rhodopsin (sR). The protein sequences of both of these proteins have been determined from the DNA sequences, hR shows 32% identity to bR (Blanck & Oesterhelt, 1987), and sR 26~/o (Blanck et al., 1989). Many (14~/o) of the residues are identical in all three proteins, including most of those known to be functionally important from the mutagenesis of bR. In addition, there is a clear indication of a greater degree of homology among amino acids that are expected to be directed inwards towards the common retinal binding site, giving further evidence that the binding site is one of the most tightly conserved characteristics of the family. A concise review pointing out the similarities between bR and hR has been written by Oesterhelt & Tittor (1989). Linear dichroism measurements have shown that the retinal absorption dipole is oriented at an angle of about 20° to the membrane plane (Heyn et al., 1977). Attempts to determine the retinal position normal to the membrane by fluorescence energy transfer, neutron diffraction and other methods have given contradictory results. However, the formation of a Schiff base b~tween the retinal and the e-amino group of Lys216, which is roughly in the middle of the hydrophobic stretch of amino acid residues that marks the C-terminal helix, means that the Schiff base of the retinal must be located approximately halfway between the cytoplasmic and extracellular surfaces of the protein. Extensive kinetic and spectroscopic analyses (Lozier et al., 1975; Kouyama et al., 1988) have resulted in the nearly universal adoption of the scheme for the photocycle shown in Figure 1. The main steps in the principal photocycle are indicated, starting from the light-adapted ground state bRs~o, which has retinal in the all-tran8 conformation. Much is known about the conformation of the retinal and protonation state of the Schiff base in each of the photochemical intermediates, from resonance Raman spectroscopy (Smith et al., 1983, 1984, 1986; Fodor et al., 1988), 13C nuclear magnetic resonance studies (Harbison et al., 1985), and analysis of the isomeric state of extraction products (Pettei et al., 1977; Tsuda et al., 1980). Fourier transform infra-red difference spectroscopy between the different intermediates has
Model for Bacteriorhodopsin Structure
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bR570
40 "¢ t~
N560
K610 /
L550
901
perpendicular to the membrane, can be considerably improved b u t we present these preliminary results now because they provide the first near-atomic resolution picture of b R in which chemically distinct groups of the protein can be discerned. We are able to present a model for the protein t h a t includes clear positions for m a n y of the most i m p o r t a n t sidechains, including nearly all the residues t h a t form the retinal binding site and those lining the p r o t o n channel.
2. M e t h o & o e
po"H
Figure 1. A scheme for the photochemical cycle of bR based on that proposed by Lozier et al. (1975). The cycle is initiated by light absorption by bRs70. A proton is released on the outside and another taken up from the inside at the steps indicated.
shown changes in aspartic acid side-chains (Engelhard et al., 1985; Eisenstein et al., 1987) and, to a lesser extent, in a tyrosine side-chain (Rothschild et al., 1986). When applied to the series of aspartic acid and tyrosine mutants, the results have suggested which of the i m p o r t a n t active site residues are p r o t o n a t e d and which are deprotonated in each of the photochemical intermediates (Braiman et al., 1988a,b; Gerwert et al., 1989). Taken together, all these d a t a form a formidable body of knowledge and have encouraged Rothschild et al. (1989a), F o d o r et al. (1988) and Lin & Mathies (1989) to suggest models for the retinal binding site and to propose mechanisms for proton pumping. However, there is still a need for a knowledge of the three-dimensional structure obtained by a direct structural method. I t is essential to prove which parts of the structure are directly involved in proton pumping and which only indirectly, either v/a more distant interactions or through their role in providing a structural framework for the groups t h a t are directly involved. We report here the results of an electron microscopic analysis, based on methods first used by Unwin & Henderson (1975), of the structure of b R in the naturally occurring purple membrane in a state close to t h a t found in the halobacterial membrane. The map represents the highest resolution so far obtained in the three-dimensional analysis of protein structure using electron crystallography to measure the amplitudes and phases of the Fourier components of the unstained structure. Although several two-dimensional crystal forms of b R are available, we have chosen to work initially on the native crystal form (space group/93, with a = 62.45 A: 1 A = 0.1 nm) because it forms the most stable andTeproducible crystals. The resolution we have reached, 3"5 ~ parallel and 10 A
Specimen preparation Purple membranes from H. halobium, prepared by the (a)
method of Oesterhelt & Stoeckenius {1974), were fused to make much larger 2-dimensional crystals using the method of Baldwin & Henderson (1984). Briefly, a suspension of 3 mg purple membrane]ml (100/tM-bR) in 0"l M-potassium phosphate (pH 5"2}, containing 6mM-octyl glucoside and 200#M-dodecyl trimethyl ammonium chloride is incubated for several weeks at 20°C. These fused membranous sheets consist of a single layer of bR molecules, which anneal into large areas of homogeneous single crystals. Because about half of the bR molecules in each fused sheet are upside down with respect to the other half, the fused membranes contain a patchwork of crystalline areas that are related to one another by twinning (2-fold axes) perpendicular to and parallel to the membrane plane. The largest untwinned areas are normally 5 to 10/~m in diameter, whereas the largest sheets are about 20 #m in diameter (Baldwin & Henderson, 1984). Since the size of the untwinned areas was generally larger than that required (5 to 7 #m) to record a cryo-eleetron diffraction pattern from a 60° tilted specimen and much larger than that required (0"8~m) to cover the field of view of an image, most of the electron diffraction patterns were from untwinned crystals, whereas none of the images so far analysed has shown twinning. For the electron microscopy, support films were prepared by evaporation of carbon on to a freshly cleaved mica surface, floating off the film on water and then lowering it gently on to 400-mesh or 700-mesh copper grids. The grids were aged in air for 4 to 8 days at room temperature before use. Specimens were then prepared by application of a drop of the fused membrane suspension to the carbon film for 30 s, removal of most of the drop by touching the edge of the grid against filter-paper, addition of a drop of 0-8% (w/v) glucose solution (Unwin & Henderson, 1975), followed by final removal of the entire drop using a second piece of filter-paper. The procedure is the same as that used by Baldwin & Henderson (1984), but some further observations might be useful. When the carbon film is fresh (less than 1 day old), it tends to be very hydrophilic. Consequently, many membranes stick to the film and the distribution looks very good; it is easy to find many large flat membranes. However, the crystallinity is usually impaired by a disordering of the lattice, which shows up as a weakening and blurring of the highresolution reflections. As the carbon film ages, it becomes more hydrophobie, and fewer membranes stick to it, but many more diffract strongly, sharply and without fall-off, presumably because there are fewer distortions through contacts with the substrate. Once the carbon film is very old (1 to 2 months), almost no membranes stick and all are washed away when the final drop of glucose is
R. Henderson et al.
902
removed by blotting. Also, on very old, hydrophobic grids the diffraction patterns from occasional membranes that do stick are again poorer. Thus, there is an optimum in the age of the carbon film at about a week. Since the fusion mixture contains detergent, the initial drop applied to the grid always spreads out and wets the carbon surface. By varying the volume of fusion mixture allowed to remain as a thin film on the grid before application of the glucose wash, it is possible to vary the composition and behaviour of the film of liquid that dries to form the final embedding medium. It is thus possible to use carbon films with differing behaviour to make excellent specimens. Basically, if the carbon film is more hydrophilic, then less fusion mixture is allowed to remain (and therefore less detergent), whereas if the grid is older and more hydrophobic, more fusion mixture is allowed to remain. In this way, good specimens (i.e. those with several membranes on each grid square, all diffracting well) could be made with a range of carbon films. Sometimes, the mica side of the carbon film gave better results than the air side. Also, ff the specimens had been prepared by leaving a larger proportion of the detergent in the final dried film (the ratio of octyi glucoside to glucose was as high as 1:15), the resulting grids did not last long. :Frequently, all diffraction would be weak or absent 24 h later. Thus, it was necessary to store the prepared grids in grid boxes in solid CO2 at about -80°C, particularly if they had been prepared with a substantial level of residual detergent. This also proved to be a convenient way of transporting the grids reliably between the different laboratories. (b) Electron diffraction A description of the electron diffraction methods is given by Baldwin & Henderson (1984). A complete set of electron diffraction data has been collected at -120°C using a Philips cold holder (PW 6599/00) on a Philips EM420 instrument, operated at 120 kV. The data scaling and merging is described by Ceska & Henderson (1990). Briefly, 150 electron diffraction patterns taken almost entirely in the form of patterns from 60° tilted specimens were scaled, merged and detwinned where necessary, to give a set of 2-9 A amplitudes complete apart from the missing cone. The coefficients describing the continuous diffraction amplitudes along the lattice lines (at intervals of 0"01 A -1) have been used as input data in the final curve-fitting using amplitudes and phases together. (c) Electron microscopy During this work, the technical side of recording the images has been most demanding and remains, with specimen preparation, as the most critical part of the work. To make progress and try to discover why the images are so much less perfect than they should be (Henderson & Glaeser, 1985), we have collaborated in using several microscopes, each of which has some advantages and some disadvantages. Of the 72 images used to determine phases, most of them (52) were recorded in Berlin on a prototype Siemens microscope, the Suleika, operating at 100 kV and liquid helium temperature (Dietrich et al., 1977). A smaller number of images (11) were included from images recorded at Berkeley on a JEM-100B operating at 100 kV and -115°C and equipped with a field emission gun. The Berkeley images were all recorded using the spotsean procedure (Henderson & Glaeser, 1985; Downing & Glaeser, 1986; Bullough & Henderson, 1987; Downing, 1988; Zemlin, 1989). Spotscan images are
recorded with a 500 to 1000 A diameter illuminating beam, which is scanned stepwise across the area to be imaged on the film. This results in a greatly decreased amount of beam-induced movement of the specimen and therefore much less blurring of the image. In spite of the much higher temperature, the best of the images from tilted specimens taken in Berkeley with spotscan were better in the tilted direction than those taken without spotscan in Berlin. The use of spotscan and the coherence of the field emission gun are clearly important for this improvement, especially for tilted images. However, by optical diffraction, the images taken at -115°C tended to have fuzzier diffraction spots than those at liquid helium temperature, which were always extremely sharp. Finally, a few images (6) were also recorded in Cambridge at liquid nitrogen temperature using a home-made side-entry cold holder (Henderson et al., unpublished results), and 2 test images were taken in Japan using the prototype Jeol 2000 SCM operating at 160 kV, with the same lens design as the Suleika. Details of the conditions used in the Berlin and Berkeley microscopes are given below. In each case, the images used in the subsequent computer analysis were carefully selected by optical diffraction, as described in Results, from a much larger number actually recorded, probably from several thousand images if we include those taken during the efforts to improve the success rate. In a good recording session (using spotscan method with an excellent grid), about half of the images obtained were good enough to include in the data processing, whilst in a bad session several hundred images might be recorded without one being good enough. (i) Liquid helium temperature in Berlin The helium-cooled superconducting cryo-electron microscope in Berlin, called SULEIKA (Supraleitender Kryoapparat) was designed by I. Dietrich and co-workers at Siemens, Munich (Dietrich et al., 1977; Lefranc et al., 1982). The main advantage of this microscope is the very low specimen temperature of -268°C, resulting in reduced radiation damage. We found by experience that a dose of 20 e/A 2 is optimal for imaging. This is perhaps l0 times greater than the optimal dose in an image of a room-temperature specimen. Some other advantages of the Suleika objective should be mentioned. (1) The magnetic field, produced by the superconducting coil and formed by the superconducting shielding, is extremely constant, like that of a permanent magnet. (2) The large pole-piece gap of 7"5 mm allows large specimen tilt. (3) The vacuum in the environment of the specimen is better than 10 -~ Torr (1 Torr ~ 133 Pa) and free of water and hydrocarbons, because the specimen is in the middle of a powerful helium-cooled "cryopump", so there is no measurable ice deposition on the specimen. The imaging procedure consists of 3 steps. In step 1, we search for a crystal. The electron optics is set to a "defocused diffraction" mode with a magnification of 500 x and a dose rate of 0.01 e A -2 s -l. The specimen is scanned by observing the image via a television camera and monitor. About 10s (0"1 e/A 2) is needed to decide whether a crystal is suitable for imaging. From time to time the crystallinity of the specimen is checked by diffraction. This requires a further 1-0 e/A 2. Before making the adjustments needed for imaging, the shutter above the specimen is temporarily closed. In step 2, we adjust the microscope. To avoid further pre-exposure, the illuminating beam is deflected to form a spot of about I pm in diameter, 2 ~m from the crystal. The deflection is adjusted to be parallel to the specimen tilt axis to avoid any focus change. Simultaneously, a 2nd
Model for Bacteriorhodopsin Structure deflection system below the specimen brings the image back to the centre of the viewing screen. Here, alongside the crystal, the defocus is adjusted at a magnification of 150,000 x and a dose rate of 10 e A -2 s -1. This is done by observing the granulation of the carbon support film v/a a television camera and monitor. When the appropriate defocus is found, the magnification is adjusted to 66,000 x where it is known there is no defocus change compared to 150,000 x . The dose rate is then set to 1"6 e A -2 s -t. For this measurement, the final screen is isolated and the beam current is measured by a pico-ampere meter. The loss of electrons, due to secondary emission, is taken into account. The microscope is now ready for recording. In step 3, the image is recorded. The shutter above the specimen is first closed, both deflection systems are switched off, and the film (Agfa 23 D 56) is exposed for 12 s. After the exposure, the defocus is measured directly on the area that has actually been photographed. For this measurement, the beam is increased to 10 e A -2 s -1 and the magnification is set to 150,000x. The film is developed in Kodak D19, full strength for 12 min. For tilting the specimen, specimen holders with fixed tilts are available for different angles. (ii) Spotscan at liquid nitrogen temperature
in Berkeley An efficient spotscan system has been implemented on a J E O L 100B electron microscope equipped with a stable, top-entry stage cooled by liquid nitrogen (Hayward & Glaeser, 1980), a field emission gun electron source, and a computer that controls lens settings and beam position (Downing & Glaeser, 1986; Downing, 1988). Conditions can be set up for searching the specimen, checking or recording an electron diffraction pattern, and focusing and recording the image. Procedures for searching and aligning the specimen are then essentially the same as for the other microscope. The main difference is that, since the J E O L 100B is not equipped with image-deflecting coils below the objective lens, focusing is carried out with the beam deflected on to an auxiliary fluorescent screen, which is mounted in front of the normal viewing screen. Thus, during focusing, the beam is deflected away from the area that is to be recorded on film. Since the deflection is generally not along the tilt axis, the focus is then adjusted by an amount that is predetermined to shift the in-focus line from the front viewing screen to a corner of the photographic film, so that the centre of the film will be at the optimal underfocus. A magnification of about 55,000 × is used both for focusing and recording the image. The high brightness of the field emission gun allows the beam to be focused to a diameter < 1000 A while still retaining the high coherence that is required for good phase contrast imaging. In practice, we usually use a beam diameter of about 1500 A, and step the beam over the specimen in a raster of 15 x 11 points to expose the full photographic film. The exposure time is typically 0"l s/spot, giving a dose of 10 to 15 e A -2 to the specimen. This short exposure time greatly reduces problems of specimen drift, but the long time to record a complete image requires excellent focus stability. In practice, we find that both the high voltage and lens current are sufficiently stable that the focus is essentially constant. (d) Image processing Approximately 20 images were included from each tilt angle range of 0% 20 ° a n d 45 °, with about 10 more at various angles in between (see Fig. 8). All images were
903
(o)
Ib)
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v
Figure 2. Illustration of the contrast variation in an image of a tilted crystal for 1 Fourier component of the structure and how it appears in the Fourier transform of the image. On the left is a function in real space and on the right its Fourier transform. (a) Variation of specimen height across image in a direction perpendicular to the tilt axis. (b) The resulting contrast for 1 Fourier component (i.e. 1 spatial frequency) of the crystal structure. See eqn (3) in Methods, section (d). The Fourier transform shows a corresponding splitting of the diffraction peak. (c) Contrast of the same Fourier component after correction in the computer by multiplication by the estimated tilt transfer function. The Fourier transform now shows the diffraction peak corresponding to the actual structure, with the correct phase. In practice, the correction is applied in the program TTBOX by convolution in Fourier transform space rather than multiplication in real space.
processed using procedures derived from those described (Henderson et al., 1986}. The image data from the untilted and slightly tilted specimens used in the earlier projection analyses (Henderson et al., 1986; Baldwin et al., 1988) are included in the present analysis without further treatment. The new image data from tilted specimens required an extension to the method of image processing as outlined by Henderson et al. (1986) and Henderson & Baldwin (1986). A more detailed explanation of the method is given below. A theoretical discussion of the correction of images for the effects of specimen tilt has also been given by Schiske (1982). (i) Correctionfor tilt For an image of a tilted crystal, the amount of defocus, and therefore the contrast, must vary across the image. Fig. 2 shows a diagram illustrating how the contrast (Fig. 2(b)) for a particular spatial frequency (e.g. one of the Fourier components of the crystal) varies with the height (Fig. 2(a)) of that part of the tilted crystal in the electron microscope. We call the function describing the contrast at a particular distance (p) from the tilt axis and at a single spatial frequency (e) t h e tilt transfer function (TTF (e, p)). This tilt transfer function is an extension of the normal contrast transfer function (CTF), which describes how image contrast varies with spatial frequency in an image from an untilled specimen. The T T F can be derived from the CTF as follows:
CTF(O) = - 2 sinT = --2 sin
AF-~--c,
,
(1)
904
R. Henderson et al.
where: is the wavelength of electrons (0"037 ,h- at 100 kV), AF is the defocus, normally in the range 0 to 10,000 A, cs is the spherical abberation coefficient (typically 2 ram) and 0 is the diffraction angle of the scattered electron beam in radians (typically 10 -2 at 3 A resolution). I n an image from a tilted crystal, AF varies with position according to the equation: AF - p t a n s + AFo,
(2)
where: AFo
is the defocus at the origin of co-ordinates, taken as the centre of the image, a is the angle of tilt measured from the plane perpendicular to the beam, and p is the perpendicular distance from the tilt axis to the point of interest. p is given by: p -- ~
sin (•-fl),
where: x, y are the co-ordinates of the point relative to the origin, Z is the angle between the line to the point and the x-axis of the digitised image (tan X = y/x), and fl is the angle between the tilt axis and the x-axis. Substitution for AF from eqn (2) in eqn (1) gives:
TTF(O, p) = - 2 sin (cp + yo),
(3)
where c and ?o are constants for given 0, defocus and tilt: C = 2~ (022 tans);
2.[
02
Thus, the image formed by a single Fourier component has a series of equally spaced, sinusoidMly varying bands of contrast parallel to the direction of the tilt axis. Such an image with this varying contrast can be treated so that all areas are restored to having positive contrast. This could be done, in principle, by multiplication of the image, pixel by pixel, by the T T F which, as a function of p, can be calculated for each position x,y on the image. This gives a new image (Fig. 2(e)), which has a contrast of (TTF) 2. The modified image has positive contrast everywhere. Also, parts that had zero contrast in the raw image and therefore contain only noise are multiplied by zero and the noise contribution removed. It can be shown that, for maximum signal to noise ratio, multiplication in the computer by T T F gives precisely the correct weight to the parts of the image with different contrast. The effect of T T F and (TTF) 2 on 1 Fourier component is shown in reciprocal space in Fig. 2. In Fig. 2(b), the actual Fourier component of the structure is split into 2 equal components, separated by a distance proportional to the number of sinusoidal variations in contrast that have occurred across the image for that Fourier component. This is because, in Fourier space, the component is convoluted with the Fourier transform of the T T F given by eqn (3). In Fig. 2(c), the actual peak is restored and 2 wings at half-height are created, separated from the correct position by the same distance as the amount of splitting in the raw image transform. Here, the split peak
has been further convoluted with the Fourier transform of the TTF. The above analysis assumes that the 2 Friedel-related diffracted beams, which combine with the direct beam to form the image, come from the same Fourier component of the structure and are diffracted at equal angles + 0 and - 0 . This assumption is valid in the current analysis. The procedure used would have to be modified if lower voltage electrons were being used or higher resolution being studied, when a pair of spots such as those in Fig. 2(b) would not be at equal distances from the ideal lattice position. An additional problem would arise if the specimen were thicker, when the two diffracted beams would also differ in their amplitude and phase. Although it has not been necessary here, the procedure could be modified to handle both of these eventualities. The convolution theorem of Fourier transform theory offers an efficient procedure for the implementation of this T T F processing. The Fourier transform of the image is convoluted with the Fourier transform of TTF, thus effectively performing the real space multiplication required to go from Fig. 2(b) to Fig. 2(c). Since the T T F is a slowly varying function, it can be described adequately by a smaller matrix (e.g. 150x 150 is adequate for a 6000 A square area of a 60 ° tilted image at 3 A resolution) than that required to sample the image in 1 A steps (this requires 6000 x 6000 pixeis). The convolution is therefore a local operation, using a much smaller part of the image transform than the entire area that would have to be treated if the processing were carried out by multiplication in real space. In earlier work (Henderson & Unwin, 1975), the inefficient real space multiplication was performed directly with an inelegant and less general trick to speed up the computation. The new procedure has been implemented in the form of 3 computer programs, TTBOX, T T R E F I N E and TTMASK, which are described below. TTBOX is the simplest of the 3 new programs. I t corrects the transform with the T T F and determines the amplitude and phase of each spot by examining a box around each lattice point. It performs the convolution correction from an input consisting of the Fourier transform of the image, and a list of values for the parameters involved in the T T F {eqn (3)). The magnification of the image is also needed in order to relate distance in the transform to true spatial frequency. Of these parameters, the spherical aberration (cs) and the wavelength (),), determined by the electron voltage (kV), are fixed and known for the microscope used; the magnification, M, the tilt angle (a) and the azimuth of the tilt axis (fl) are known approximately when the image is taken and can be determined accurately from the foreshortening of the lattice para~meters found in the image transform (Shaw & Hills, 1981); the defocus (AFo), which in general is different for spots in different parts of the transform due to astigmatism, is a function of 3 parameters, AFt, AF2 and ~b, whose values have to be determined during the analysis: AFo = AF 1 cos 2 ( ~ b - ¢ ) + A F 2 sin 2 (~h-¢},
(4)
where AF t and AF2 describe the amount of defocus in the centre of the image in 2 orthogonal directions. ¢ is the angle specifying the direction of AF t relative to the x-axis of the digitized image. Eqn (4) gives the value of AFo that applies to a Fourier component whose direction in the transform is at an angle ~ to the x-axis of the digitized image (Zemlin et al., 1978). The output of TTBOX consists of a list of amplitudes and phases for the Fourier components of the crystal, fully corrected for tilt, defocus and astigmatism, and ready to be merged with data from other images. There
Model for Bacteriorhodopsin Structure are also several useful diagnostic graphical and tabular printouts, which enable the experimenter to judge whether an image is good and whether the processing has been optimized. The amplitude and phase at each lattice point are accompanied by a calculation of the local value of the r.m.s, background in the transform taken from the perimeter of a box surrounding the lattice point, together with an estimate of the resulting error in the phase. We have found it useful to define, for each spot, a parameter indicating spot quality, IQ = 7 (B/A), where A is the background corrected amplitude of the spot and B is the background near the spot. Thus, a spot with IQ = 1 is very good, with signal to noise ratio greater than 7"0, whereas a spot with IQ = 7 is very weak, with a signal to noise ratio of 1"0. We use IQ = 8 to describe a spot whose amplitude is above background but by an amount less than B, and IQ = 9 when the Fourier transform value at a lattice position is less than the r.m.s, background (i.e. A is negative). In the Fourier transform of an image without any specimen present, 65°/o of the data have IQ = 8 or 9 because of the x e -x shape of the distribution of noise (Hayward & Stroud, 1981). (ii) Correction for spatial distortions The above has described how the T T F processing is carried out, but real images also contain spatial distortions. The most important are those arising from stretching and bending of the crystal or the presence of cracks, defects or dislocations of the lattice due to crystalline defects. In the spotsean images, drift over the long period of exposure sometimes produced substantial shifts of the different areas that make up the image. These distortions, as well as those arising from pincushion and spiral distortions of the electron microscope, can be removed by the application of real space-correlation methods (Henderson et al., 1986) based on methods similar to those used previously by others (e.g. Crowther & Sleytr, 1977; Frank et al., 1978; van Heel & Hollenberg, 1980; Crepean & Fram, 1981; Frank, 1982; Saxton & Baumeister, 1982). (iii) Interdependence of defocns and spatial
distortion corrections For the tilted specimens, the problem is that the T T F correction in reciprocal space as well as the unbending correction in real space are both 6ssential and interdependent. After some preliminary experiments, in which the order of the application of the corrections and the amount of iteration was varied, we settled on the scheme shown as a flow diagram in Fig. 3, in which the unbending and the T T F correction were both improved in a cyclical manner. Two further extensions of the TTBOX program were needed in order to achieve this. The functions of these 2 programs, T T R E F I N E and TTMASK, are described below. T T R E F I N E allows the 3 defocus parameters (AFI, AF2 and ~b in eqn (4)) on which the T T F depends to be refined. T T R E F I N E is an iterative least-squares refinement program that maximizes the sum of the TTF-correeted amplitudes of all the spots within a chosen resolution range. The quantity to be maximized is ~'~.A2, where A denotes a TTF-corrected amplitude: A =
.
6A.
opl
apl+
6A . - ap2+
op2
~A
.
- ap3,
op3
(5)
905
where Pl, P2 and P3 are the parameters AFI, AF2 and ~b, A 0 is the TTF-corrected amplitude based on current parameters, and 5A/Spl, ~A/Sp2 and 6A/~p3 are the partial derivatives of the TTF-corrected amplitude with respect to the parameters. These are produced by convolution of the image transform with the transform of the partial derivative of the T T F with respect to the parameters. For example, 5A[$pl is calculated using the transform of 5(TTF)/Spl, which is worked out, pixel by pixel, by differentiation of eqn (3). Note that in eqn (3), ?o is a function of AFo, and AFo is specified by the parameters as given in eqn (4). Thus, T T R E F I N E calculates 3 more convolutions in addition to that carried out by TTBOX. The improvement of the defocus parameters is normally continued until the refinement converges. The T T R E F I N E step, used at the end of each pass, can consume a lot of computer time (4 times the number of convolutions and many cycles compared to the single convolution in TTBOX). In the beginning, we tried to estimate the midimage defocus by optical diffraction so that refinement could begin near the correct value. Later, with experience, we simply guessed a reasonable value and tried a series of different starting points, allowing the refinement program to show which defocus values gave the largest number of good spots (those with low IQ) and the highest average amplitude. Typically, the program is able to converge from defocus estimates that are within 1000 A of the correct values. The selection of a starting point further from the correct value than this would cause the refinement to go in the opposite direction and end on a smaller maximum. This is because the T T F is a sinusoidal function and repeats itself exactly at a different height for each spot. 0nly for a single value of mid-image defocus, however, are all spots treated correctly. Care is therefore needed in using T T R E F I N E . Fortunately, in the later steps of merging data from different images, there are many checks that the correct defocus has been determined. T T R E F I N E can also refine the tilt parameters a and fl, but in the analysis reported here these have been left at their initially determined values, which are sufficiently accurate. The program TTMASK, used at the beginning of second and subsequent passes, reads in the Fourier transform of an image and writes out another Fourier transform, corrected for tilt, defocus and astigmatism. I t also carries out masking using circular or square apertures around the position predicted for each spot and leaves the whole area of the TTF-corrected transform inside the mask rather than just the single amplitude and phase at the predicted position, as is done by TTBOX. This allows the data that describe the image distortions to remain in the TTF-corrected transform, whilst removing noise from between the spots. (iv) Flow chart The 3 programs TTBOX, T T R E F I N E and TTMASK are used in the procedure as shown in Fig. 3. The other steps in the scheme use the established set of programs for correcting untilted images for distortions (Hendemon et al., 1986). In the 1st pass through the scheme, the image is treated as if it were untilted and the steps taken are shown on the left-hand side of the diagram. The 1st pass normally resulted in the successful unbending of a strip of the image that extended across the full width in a direction parallel to the tilt axis, but was more limited in the perpendicular direction. For 45 ° tilted images, only about 30% of the area was treated successfully in the 1st pass. The values of the defocus and astigmatism parameters
R. Henderson et al.
906
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List of amplitudes and phases i Figure 3. A flow diagram showing the computing steps in the processing of images from tilted specimens. Data sets are enclosed in boxes; programs are in bold type. Light shading behind the program name indicates that the program was developed earlier for the processing of images from untilted specimens; black shading indicates a new program developed for the work with tilted specimens. Broken lines indicate the transfer of refined parameters; full lines indicate data flow. AF1, AF2 and ¢ could then be determined using TTREFINE, and these values used as input to TT~J~SK in the 2nd pass of unbending, as shown on the right-hand side of Fig. 3. The 2nd pass was carried out using the masked TTF-corrected transform of the raw image together with the TTF-correeted Fourier transform of a reference area taken from the unbent image of the 1st pass. By using a tight mask for the Fourier transform of the unbent image,
a very highly averaged reference area is obtained, whilst a generous mask for the Fourier transform of the raw image enables all the distortion information to be included in the analysis. In subsequent passes the procedure cycles as indicated on the right-hand side of the diagram, as the unbending and the accuracy of the defocus parameters improve together. Because the contrast varies so much across the image from a tilted specimen (see Fig. 4(d)), it
Model for Bacteriorhodopsin Structure was not possible to use the weighting procedure described by Baldwin et al. {1988) in the new scheme for tilted specimens, although it would be desirable to do so if it were possible to devise a procedure to pick out which areas of the image were best and therefore should receive the highest weighting.
{e) Parameter determination and data merging The Fourier transform of a 2-dimensional crystal consists of a series of lattice lines continuous in the zdirection. The electron diffraction intensities had been merged previously and intensities fitted to curves along each of these lattice lines using damped sine functions (Baldwin & Henderson, 1984; Ceska & Henderson, 1990). The fitted curves consisted of intensities sampled at intervals of 0-01 A - I along z*. They were converted to amplitudes using the program TRUNCATE (French & Wilson, 1978) from the CCP4t program suite. TRUNCATE converts intensities to amplitudes, taking into account the correct treatment of negative intensities. Phases from the new images were analysed and compared using the program 0RIGTILT. This program enables the phase origin, and/or crystal tilt and/or beam tilt parameters of individual images to be refined as well as data from many images to be merged. Phases from each image were first compared with the phases produced by earlier averaging of 6 A 3-dimensional data from 3 crystal forms at room temperature (Tsygannick & Baldwin, 1987). At 6 A resolution, a knowledge of the beam tilt (Henderson el al., 1986) is not necessary, so this preliminary treatment of the phase data simply involved bringing the data from all the images to the common crystallographic phase origin. Imperfect alignment of the illuminating beam affects the high-resolution components of images (Zemlin et al., 1978). The amount of beam tilt must therefore be determined and its effect corrected for. The determination of beam tilt (2 parameters specifying its amount and direction) for images of untilted and slightly tilted specimens had been done (Henderson et al., 1986; Baldwin et a/., 1988). The determination of beam tilt for the images from tilted specimens required an iterative procedure and was carried out in 2 stages, first for all the 20° tilts, then for the 45° tilts. For the 20° tilts, a merged, list of phases was first produced with the 20° image beam tilts set to zero b u t including the previous correctly treated untilted data. This merged list was then used as the reference for preliminary refinement of beam tilt .by comparing data from each image against data from all the others, excluding itself. Second and subsequent merges, followed by further beam tilt refinement resulted in the iterative convergence of beam tilt and phase origin to, in most cases, unambiguous values with convincing phase residuals, which showed good agreement at resolutions out to 3"5 A (see Table 2). The 45° tilts were then included with preliminary beam tilts derived by comparison with the 20 ° merged data, followed again by iterative refinement. Ten cycles of iterative merging and beam tilt refinement were carried out in this way. The merged list of phases (1.7,922 phase measurements from 72 images including all data with IQ values between 1 and 8) was then combined with the list of amplitudes from electron diffraction and used as input to the program
~f CCP4 is the Collaborative Computational Program in Protein Crystallography.
907
LATLINE (Agard, 1983). LATLINE fits amplitude and phase curves to the non-uniformly sampled data on each lattice line. One of the constraints used in the fitting algorithm is that no electron density should be found outside a specified membrane width profile, set in our case as - 2 1 A to +34 A. This 55 A width is greater than the 45 A minimum stacking distance (Henderson, 1975) seen by X-ray diffraction of dried membranes and was chosen to avoid the possibility of erroneous truncation of density. The profile is centred on a phase origin that is about 6 A from the middle of the membrane because of the asymmetric mass distribution. Each amplitude and phase is accompanied by its own standard error, a, and these were used to calculate l/a 2 weights, which are used by the LATLINE program to calculate the standard errors of the fitted values as well as the best fit. The amplitude data were already adequately sampled (Ceska & Henderson, 1990) at 0"01 A -1. I f new phase data for a lattice line were not measured over any region wider than 0"02 A-1, the fitting of the curve was truncated to the continuous region that was well sampled. The output from LATLINE is then a set of amplitudes and phases that represent the best estimates of the structure factors from all the data. Note that this method of merging makes no use of the image amplitudes, and assumes that the amplitudes and phases are derived from precisely the same structure, even though they are measured by different methods. There is little doubt that these are good assumptions, since the electron diffraction amplitudes are much more accurate than the image amplitudes, and the high correlation coefficients obtained between image and electron diffraction intensities (see Results) shows that, at least for the best images, the structures being observed are the same. Having obtained a complete set of merged amplitude and phase data, 2 final checks were carried out. First, the tilt axes and tilt angles of all the images were refined by minimizing the phase error by comparison with the smooth data from lattice line curves. This was done using 20 to 6 A resolution data only, where the phases are most accurate. Second, the beam tilt on each image was now refined against the merged curves as a final check and to get the most accurate values. These curves were then used to calculate a Fourier map as described below. A 2nd set of curves was fitted with data modified by using individual weights for each spot obtained from calculations of the resolution-dependent fall-off in intensity for 2 directions on each image {Le. parallel and perpendicular to the tilt axis). These individual image weights were used to modify the 1/a2 weights for a 2nd run of LATLINE, and a 2nd set of curves used to calculate a 2nd map. However, the 2 maps are very similar. The weighted map was used in all subsequent work. if) Calculation of map, interpretation and model building Following the calculation of 3-dimensional amplitudes and phases sampled at 0"01 .h.- 1 along the lattice lines, the data were converted into LCF (Labelled Column Format) data files used by the CCP4 crystallographic program suite. All further calculations could then be carried out using standard crystallographic programs. The standard errors on the phases calculated by LATLINE were converted into figures of merit and used as weights in the calculation of the Fourier map. Interpretation of the map and model building into the density were aided by stacks of transparent contoured sections {minimaps) and the graphics program FRODO (Jones, 1978}. Frequent use of the regularization option in FRODO
908
R. Henderson et at.
allowed the model to be constrained to a-helices where apropriate. The R-factor between calculated and observed amplitudes, using all electron diffraction amplitude data to 3"5 A resolution and 60° tilts, was 45% for the initial model, which included 10 lipid molecules/asymmetric unit. A final pass of the raw co-ordinates through 50 cycles of Hendrickson-Konnert (Konnert & Hendrickson, 1980) refinement produced a model with good stereochemistry and an improved R-factor (35 % for all data to 3"5 h). 3. R e s u l t s
(a) Electron microscopy and assessment of data Images of tilted specimens, recorded on any of the microscopes used, were examined by optical diffraction. Good images were immediately obvious, showing strong sharp diffraction spots extending to high resolution. By contrast, bad images frequently showed very few diffraction spots. Between these two extremes, there was a great variety of images with all sorts of defects: a thin band of spots due to specimen or image movement during the exposure; very fuzzy spots everywhere, indicating disorder of the specimen during specimen preparation; variability of resolution over different areas of the same micrograph; strong low-resolution spots with nothing visible beyond 10 A. To develop a simple criterion that would allow us to decide which images should be subjected to computer analysis, we endeavoured to estimate the resolution parallel and perpendicular to the tilt axis, and counted the total number of unique spots visible in the optical diffractometer. This latter criterion, the total number of spots visible, turned out to be the most useful, although some images showed such an obvious indication of one good direction and one bad direction, that it was easy to take this into account. However, attempts to make more exact estimates of resolution at the optical diffraction stage turned out to be less valuable. Some of the images that had looked less promising at the optical diffraction step, turned out to contain good highresolution data after computer processing. As a result, we resorted to the procedure of selecting the best images, but also including many that looked less good at the stage of optical diffraction. "This procedure ensured that we did not exclude any good images from the computer analysis, but resulted in the inclusion of some images with very weak highresolution spots. Lattice and image distortions are taken care of by correlation analysis. In Figure 4, for an image of a 44 ° tilted specimen, we show the maps of the correlation peaks obtained during the first pass ((a) and (b)) of analysis and during the last pass ((c) and (d)), following the procedure described in Figure 3 and Methods, section (d). During the first pass, no correction for defocus has been made but, since most of the image is slightly underfocused, an approximate correlation map is still obtained. The bottom left corner of the image shows the lower contrast that is expected for the part of the image that is closer to
focus. In images of more highly tilted specimens such as this, the band of zero contrast, where the specimen height brings a region of the image precisely into focus is often wighin the processed area, together with an area of overfocus on the other side of the band. In Figure 5, we show in resolution zones the average quality of the spots in the Fourier transform of an image after application of the full correction for distortions and defocus. In this case, the image was recorded from a 41 ° tilted specimen using the spotscan procedure. In all zones, and in directions both parallel and perpendicular to the tilt axis, the average diffraction spot is welt above the mean background. Clearly, the lower-resolution data are best and at high resolution, the data parallel to the tilt axis have a higher signal-to-noise ratio than the data perpendicular to the tilt axis. Figure 6 shows, for the same 41 ° tilted specimen as in Figure 5, a representation of all the spots out to 3"5 A and their strengths, indicated by their size and IQ value (1 is best, see Methods, section (d) for definition). Figures 5 and 6 demonstrate the presence of many strong spots in the corrected transform of the unbent image. Further proof that the data obtained from these images arises from the same structure that we see by electron diffraction is shown in Table 1, which shows the correlation coefficient between the intensities (amplitudes squared) of the diffraction spots in the image and the corresponding intensities from the merged electron diffraction patterns (Ceska & Henderson, 1990). The correlation is more than 0"47 at all resolutions and in all directions, except in the intermediate resolution zone in the tilted direction, where the diffraction is always very weak. In some images from 20 ° tilted specimens, the correlation was more than 0"80 in all zones and all directions. However, other images we have included from highly tilted specimens were not as good as this. In the worst cases, correlation coefficients dropped to near zero in the highest resolution zone. In nearly all images, the spots perpendicular to the tilt axis in the higher-resolution zone were the weakest, as would be expected if they were affected most by lack of specimen flatness and beam-induced specimen movement perpendicular to the support film. In all, from about 50 images of tilted specimens that were subjected to computer analysis, about 30% showed peaks greater than 10 in plots similar to those in Figure5 and correlation coefficients Table 1
Correlation coe~cients between image intensities and data from diffraction intensity curves for image 51056 Position relative to tilt axis Resolution (A)
Parallel
Diagonal
Perpendicular
oo-7 7"0-5"5 5"5-3"5
0"98 0"95 0"92
0.96 0"95 0"66
0"91 O"17 0"47
Model for Bacteriorhodopsin Structure fo}
(c)
909 (b)
(d)
Figure 4. The correlation peaks found in a search of the correlation map of image 20945 showing the positions and heights of the correlation peaks at each of the 20,000 unit cells of the crystal present in this image. The image is from a 44° tilted specimen. In the' 1st pass ((a) distortion vectors ( x 10) and (b) peak heights), a large reference area from the centre of the filtered image is used to calculate the correlation map. The reference area can be seen in (b) as the area of highest correlation. In the final pass, ((c) distortion vectors ( x 10) and (d) peak heights), a smaller reference area is used, which is derived from the average of the entire area of the image after corrections for distortions and defocus as defined in the penultimate pass. In this case, maximum correlation peak height is found at the top of the image where the undeffocus is greatest, and almost zero contrast is found at the bottom of the image where the image is almost exactly in focus. Because of the smaller reference area, finer distortions can be seen in (c) than in (a).
(Table 1) greater than 0-2 at the highest resolution, 30~/o showed diffraction peaks detectably above those expected from an image consisting of pure noise, and the remainder, in effect, contributed diffraction information only out to 5"5 A resolution. Phase d a t a from all images were then merged together using the program O R I G T I L T to determine the phase origin and beam tilt parameters. In earlier stages of processing, only the amplitudes of the diffraction spots were used as diagnostics to check t h a t the correct tilt axis and tilt angle were
being used and t h a t the* determination of defocus was optimal: At the stage of running the program O R I G T I L T (Table2), agreement between the phases of each new image and the phases from other images gave a final check t h a t everything had been done correctly during the processing. The phase agreement is shown for the best 45 ° image in Table 2. The fact t h a t all the residuals, even at the highest resolution, are significantly lower t h a n expected for r a n d o m phases (90 ° ) means t h a t . t h e phases from the image itself are good, and t h a t the
R. Henderson et al.
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Figure 5, Areas surrounding the diffraction spots in the Fourier transform of the distortion and defocus corrected image 51056, divided into resolution bands and analysed separately for spots parallel and perpendicular to the tilt axis. The image is from a 41 ° tilted specimen. The tilt axis is inclined at approximately 65 ° to the horizontal X-axis. The plot is centred on the spots and calculated by averaging the diffraction intensity of all 38 spots in each plot at 7 A, all 27 spots at 5-5 A, and all 96 spots in each 3"5 A plot. The peak in the centre of panel 1 (top left) is 2065. Thus, in this image, the average diffraction spot in the highest resolution zone perpendicular to the tilt axis is above background by 1"5 x background. At 7 A, spots are from 150 x to 300 x background. The wings expected from the contrast stripes in the image can be seen best near the edge of the box in the 5-5 A plot for the stronger spots from the direction parallel to the tilt axis. Below 7 A resolution, the wings show up as shoulders to the main peak, and at 3-5 A the wings are off the edge of the plots. combined data from the other images with which they are being compared are good. Thus, Table 2 is a final indication that high-resolution phases in three dimensions have been obtained successfully. Following the merging of phase data into a set of origin-corrected and beam tilt-corrected measure-
ments, s m o o t h curves were fitted along lattice lines to the combined electron diffraction amplitude and image phase data using the program L A T L I N E (Agard, 1983). S o m e typical plots of amplitude and phases after L A T L I N E are s h o w n in Figure 7. Error estimates on the fitted curves are also obtained. At
tJ
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Figure 6. Plot showing all spots observed above background in the transform of image 51056 after full corrections. The circles show resolutions of 7, 5-5 and 3"5 A. The size of the symbols is proportional to the strength of the spot. Numerical grades (IQ value) are shown for the strongest spots. Grades 1, 2 and 3 are highly significant and give accurate phases. The general trend for spots near the tilt axis to be stronger and spots perpendicular to the tilt axis to be weaker is clear.
Table 2
Phase residual for image 51056 Res rain (A) 100"0 14-0 10"0 8"1 7"O 6"3 5"7 5"3 5"0 4-7 4.4 4-2 4"O 3"9 3"7 3"6 Overall
Res max (A)
Phase residual (degs.)
Number of phases from spots with IQ<~7
14'0 10"0 8"1 7'0 6"3 5"7 5"3 5"0 4"7 4.4 4"2 4~) 3"9 3"7 3"6 3"5
13"1 7-2 15-5 16-8 2@9 2~7 35"0 28~) 40.2 38"9 28"6 58"4 52"3 45"7 36"9 19-2
19 21 19 19 15 18 13 11 15 11 11 8 4 8 4 I
26-3
197
Note that this is only one of 72 images, and only spots with amplitude greater than a certain value (given by IQ<~7) are used in the phase comparison. Fig. 8 gives an idea of the total extent of the phase data.
R. Henderson et al.
912
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Figure 7. Lattice line data. Plots of amplitudes (AMPL) and phases together with the curves produced by weighted least-squares fitting using the program L A T L I ~ (Agard, 1983). Every phase measurement is plotted either as a large circle (IQ 1 to 4) or a small circle (IQ 5 to 8). Amplitude measurements are derived from earlier data (Ceska & Henderson, 1990). For these 4 lattice lines, selected from 143 used in the map, it can be seen that phases are most accurately determined when the amplitude is high and least accurately when it is low.
this stage, the weight applied to each phase measurement in the input to curve fitting was a function both of the strength of the spot in the image transform (weight oc 1/a 2) and of the quality and strength of the n e a r b y spots from the same image (see Methods, section (e)). This allowed weak spots on good images to contribute more than spots on poorer images t h a t were known from neighbouring spots to have a very low information content. This weighting scheme eliminated subjective judgements of good and bad images and produced a smooth transition both within and between images. Figure 8 summarizes the e x t e n t in three dimensions of the amplitude and phase d a t a used. Because
images were collected mainly near three tilt angles (0 °, 20 ° and 45°), the distribution of experimental phase measurements is not uniform, as shown in Figure 8 where density of shading represents density of sampling by the images used. Nearly all the Fourier components determined by curve fitting up to 20 ° tilt are well measured, whereas those up to 45 ° are noisier with greater phase errors in the fitted curves. I f all phases up to 45 ° were well measured, then 71 °/o of the Fourier components in the threedimensional transform would have been determined. However, some lattice lines did not have phase d a t a t h a t were sufficiently well sampled, because some parts of the three-dimensional
Model for Bacteriorhodopsin Structure 0.3
(b) Resolution o/the three-dimensional map
nt extent of ~s (imoges)
o~
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0
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-0.3
913
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Figure 8. Summary of the full extent of measured amplitude and phase data, plotted as radius in the X* Y* plane versus Z*. The density of shading denotes density of sampling of the phases in the separate ranges of data from 0° (2"8A), 22° and 45° (3-5 A) tilted specimens. The limit of the older 6 A phases data from 3 crystal forms at room temperature (Tsygannik & Baldwin, 1987)_ is also indicated, as are the 60° tilt and 3-0 A limits of the electron diffraction measurements. The shape in the corner shows the point response function corresponding to the distribution and estimated accuracy of our phase data. The width of the point response function at half-height is 2"7 A in the X, Y plane and 5"6 .A in the Z direction; point features in the structure separated by a distance equal to the halfwidth would be just resolved. These half-widths correspond to effective Fourier cut-off resolutions of 3"5 A and 7-8 A, respectively. (If we had a complete, accurate phase data set from 45° tilts, the half-width in the Z direction would be 3"9 A, and the corresponding resolution would be 5"5 A.)
The Fourier cut-off resolution was 3"5 A in the horizontal direction but, because of the limited tilt range of the data collection, the highest resolution Fourier term that is possible in the vertical direction corresponds to 5"5 A. A section through the map just below the middle of the membrane, plotted with ten different contour levels, is shown in Figure 9. The entire map in 7 A slices is shown in Figure 10 contoured only at the second level. One of the 7 A slices is shown with the model superimposed in Figure 11. The resolution achieved can be seen by looking at several profiles through different peaks in the map (Fig. 12). The peaks have the expected shape. Figure l2(a) shows a horizontal profile through the largest peak in the map, which is the fl-ionone ring of retinal as explained below, giving a good estimate of horizontal resolution. Figure 12(b) shows a very similar profile through one of the aromatic side-chains (Tyr185). Figure 12(c) shows a vertical profile of the retinal peak, illustrating the lower vertical resolution as well as the presence of almost resolved features about 7 A above and below it. Finally, in Figure 12(d), we show a quite independent way of illustrating the vertical resolution. Purple membrane embedded in phosphotungstate has been studied by Ceska & Henderson (1990), who collected a full set of three-dimensional diffraction data to 3"0 A resolution. A three-dimensional difference map between phosphotungstate-embedded and native purple membrane, using the new phases, shows several large peaks on the two surfaces of the membrane where the tungsten clusters bind. Since the heavy-atoms are small dense features, their profiles can be used to give a direct indication of resolution. The widths of the phosphotungstate profiles confirm the native vertical resolution seen in Figure 12(c). Thus vertically, peaks 7 A apart are just resolved. The full width at half-maximum of each peak is about 2"5 A horizontally and 7 A vertically. These figures imply an effective Fourier resolution of 3"5 A horizontally and I0 A vertically. (c) Interpretation of the three-dimensional map
transform are intrinsically weak, and others were poorly sampled by the random distributions of tilt axes and angles used. Consequently, the curve-fitted data produced 2700 Fourier components with significantly measured phases, representing about half of the data in a full three-dimensional sample: 29 % of reciprocal space representing the 45 ° missing cone is not sampled at all in this set of data. Figure 8 also shows that electron diffraction amplitudes have been measured up to the higher tilt angle of 60 ° (Ceska & Henderson, 1990) representing 87 % of amplitudes. The unphased amplitudes, some of them at exactly the resolution where the 5 A axial diffraction from a-helices is strong, could not be used in the calculation of the map but will be used in the refinement of the model.
The map shows numerous features that are well resolved from the axes of the seven helices. These must be the side-chains of bulky aromatic amino acids such as Phe, Tyr and Trp as well as the retinal. In particular, the densest peak in the map (Figs 9, 12(a) and (c), and 13(a)) is in exactly the position expected for the p-ionone ring of the retinal, based on the position of the ring label peak in the neutron diffraction difference map (Heyn et al., 1988). Vertically, the peak is about 6 A below the centre of the membrane judged from the positions where density begins on the top and bottom sections of the map. There is therefore no doubt that this peak is the /Y-ionone ring. Above and below the fl-ionone peak (height 10 contours) are almostresolved peaks of half the height (5 contours). Most
R. HendersoT~ et al.
914 ....
>)'
Figure 9. A section through the map parallel to the membrane plane at the level of the fl-ionone ring of the retinal, just below the centre of the map. There are 10 equally spaced contours with the maximum density in the ring of the retinal.
(o)
(g) . . . .
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Figure 10. Each panel ((a) top, (g) bottom) sh~ws a 7 A thick slice through the map parallel to the membrane plane, contoured at a level of 20% of the maximum density. There are 7 slices from the top to the bottom. In the section below the central one, the retinal ring can be seen together with several resolved side-chains. This same section is shown with the atomic model superimposed in Fig. 11.
i
916
R. Henderson et al. Number of contours -10
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Figure 12. Profiles through several identified features to estimate horizontal and vertical resolution. (a) The retinal ring in the horizontal direction. (b) Tyr185 in the horizontal direction. (c) The retinal ring in the vertical direction. (d) Phosphotungstic acid embedded-glucose embedded difference map peak in the vertical direction.
of the other well-resolved peaks are also of height about five contours, whereas the density at the centre of the helices on average rises to eight or nine contours. Noise was one to two contour levels as judged by looking at the map in regions above the ends of the helices but within the membrane profile. We looked first for density connecting the helices at the top and bottom of the map. Regarding the connectivity at the top (Fig. 10), we find that the density can b e followed upwards from each helix until the density from an adjacent helix either merges with it, or the densities from the two helices get so close together and at the same time so far from other helices that the connectivity of the~polypeptide in each region is in little doubt. Similarly, for the bottom sections of the map, where much larger and denser features are present that are quite clearly not part of the seven vertical helices, the density seems to connect pairs different" from those joined at the top. While no connection in the map is unequivocal/the fact that the six indicated connections are self-consistent confirms that the seven helices have the connectivity shown in Figure 14. The helices without a connection, one each at the top and bottom of the map must then be the amino and carb0xyl termini. It is known that the amino terminus is at the bottom of the map, and corresponds to the extracellular surface from biochemical sidedness experiments (Gerber et al., 1977) and electron diffraction of membranes with known orien-
tation (Henderson et al., 1978; Hayward et al., 1978). Hence, the helices in Figure 14 can also be labelled A to G according to their position in the sequence as shown. The connectivity and helix assignments are the same as those suggested previously (Engelman et al., 1980). We can then look to see whether the positions of the bulky aromatic side-chains in the amino acid sequence correspond with the positions of the features that are well resolved fromthe main density of the helices. We looked first gtj helix F where the residues Trp182, Tyr185 and Trp189 are the only aromatic residues in the bottom half of the helix near the retinal ring. The two tryptophan residues are seven residues apart and therefore must be almost vertically above one another separated by 10'5A; Tyr185 must be about 60 ° anticlockwise from the tryptophan residues and halfway between them vertically. If we orient the helix so that the nearly unresolved features above and below the retinal ring represent the indole moieties of the tryptophan residues, there is a density in precisely the right place for Tyr185, which would place the tyrosine side-chain more or less parallel to the aliphatic part of the retinal. Figure 13 shows stereo views of our interpretation of these two features in the map. In Figure 13(a), the fl-ionone ring of the retinal is barely resolved from the side-chain of Trp189 below it. The side-chain of the residue above the ring shows up as a shoulder in the density. Figure 13(b) also shows that the density in helix F that we assign to Tyr185 extends downwards and becomes closer to the adjacent helix G, suggesting the presence of another side-chain from helix G at this lower level. From the sequence, Phe208 is the only aromatic residue in the lower half of helix G. Our interpretation of Figure 13(b) is therefore that the side-chains of Tyrl85 and Phe208 appear as a single elongated peak joining the two adjacent helices together. One consequence of the assignment of the side-chain of Phe208 to the lower half of the density shown in Figure 13(b) is to enable Lys216, connected to the retinal, to be positioned 12A higher than Phe208 and about 80 ° anticlockwise from it. This puts Lys216 in the correct position to form a Schiff base with the aldehyde of the retinal, assuming the retinal to be tilted at about 20 ° to the plane of the membrane, as shown by linear diehroism measurements (Heyn et al., 1977). Similar, clear-cut interpretations of the densities in the lower halves of helices C and E can be made. In each case, there are aromatic side-chains in the sequence and corresponding densities in the map; Trp137 and Trp138 for helix E, and Tyr83 and T+p86 for helix C, as well as a few others. 'Helix B contains rather a lot of bulky residues and the map contains a number of peaks. Consequently, there were two possible orientations that fitted the density, with either Met56 or Tyr57 directed towards the environment of the Schiff base. Since Tyr57 is conserved in all the members of the bR family (see Fig. 19) and is known from mutagenesis to affect the rate of reconstitution of
Model for Bacteriorhodopsin Structure
917
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Figure 13. Stereo view of 2 parts of the map to illustrate the poorer vertical resolution and the results of our efforts to interpret the map. (a) The retinal ring viewed parallel to the membrane showing Trp189 and Trp86. (b) The unresolved density for Tyr185 from helix F and Phe208 from helix G.
apo-membrane with retinal (Mogi et al., 1987), we decided that the second orientation, with Tyr57 pointing inwards, must be correct. Helix A, which is knowr~ to be less important in the functioning of bR (Khorana, 1988), contains two tryptophan residues at positions l0 and 12. We fitted these, as well as Met20, Tyr26 and Phe27, into features that seem unique and that result in an orientation in close agreement with that determined by Popot et al. (1989) using neutron diffraction of
membranes containing leucine-labelled helices A and B. The remaining helix D contains no bulky aromatic side-chains and the map showed no clearly resolved densities. As a result, the orientation of this helix is the least certain part of our interpretation. We simply placed the helix in the density to maximize the correlation of the positions of the five glycine residues with the apparent absence of bulges along the main helix density, and with. A s p l l 5
918
R. Henderson et al.
(b) Figure 14. Overall chain trace. (a) A diagram showing helices as solid rods; (b) ribbon diagram following the backbone of the polypeptide. Helix A, on the left, is connected to helix B at the top; helix B is connected to helix C at the bottom; helix C to helix D at the top; helix D to helix E at the bottom; helix E to helix F at the top; helix F to helix G at the bottom. The N terminus (nt)is at the bottom at the extracellular surface; the C terminus (ct) is at the top at the cytoplasmic surface. Both diagrams show the retinal and its connection to Lys216. oriented inwards, towards and above the retinal ring. This orientation also brings the other two conserved residues in helix D (Fig. 19), Metll8 and Gly122, into the environment of the retinal ring. Thus, the positions of the residues of helix D, while plausible, are not determined with certainty. Fortunately, the helices forming most of the environment of the retinal, B, C, E, F and G, are the most unambiguous. Figure 11 shows the superposition of the atoms on the density in the 7 A slice of the map at the level of the retinal, according to the interpretation given above, after model building and refinement. The final part of the interpretation that gives us confidence comes from the consequence of the identification of well-resolved features in the map with aromatic side-chains in the sequence. We can build each helix upwards and downwards from the assigned aromatic residues and ask whether the remaining lengths of polypeptide chain are consistent with the observed hydrophilic inter-helix links, some of which are small and tight (e.g. CD)
and others of which are much longer (e.g. BC). Considering that the vertical resolution is poor and therefore that the exact conformations of the links are harder to see, there is an excellent correspondence within one or two residues between the observed density in each link and the length of sequence expected from side-chain assignments. In particular, the weaker densities for the three links at the top correspond to extended polypeptide chain, whereas the much denser features at the bottom of the map have bigger stretches of polypeptide to fit into them. Indeed, all three Iinks at the bottom have a density and length that suggest that short stretches of horizontal ~-helix are required to fit the required number of amino acid residues into the density that is present. The remainder of the map was interpreted by building up and down from identified regions using the bulges t h a t protrude from the helices to position the side-chains from smaller amino acids. This served to keep the helices in register, and should result in a correct interpretation, except where there
Model for Bacteriorhod~sin Structure
919 CYTOPLASM
A
B
C
D
E
F
EXTRACELLULAR G
Figure 15. A view of each helical section of the main-chain backbone. The cytoplasmic side is at the top. Clear kinks in helices B, C and F are seen at the positions of Pro50, Pro91 and Pro186. Helix G also seems to be bent near Lye216, whereas the other helices A, D and E are relatively straight. The viewpoint for each helix has been selected to show best the deviations from straightness. Helices B, C and D are viewed parallel to Y. Helices A, E; F and G are viewed parallel to X*. Fig. 9 shows these directions with respect to the 7-helix plan view.
is a possibility of a kink such as might exist at the proline residues in helices B, C and F. Fortunately, this does not seem to be a problem, and the bulk of the structure is unambiguous, including most of the retinal binding site and the residues that line the proton channel near Asp85 and Asp96. No density is seen in the map after residue 225 at the C terminus, nor before residue 8 at the N terminus. (d) A model of the structure of bR The first problem we had in building the detailed model into the density was the conformation of the retinal. The map shows the position of the Schiff base approximately in the centre of the molecule and the ring about 5 A lower towards the extracellular surface, but the resolution cannot show the methyl groups. This gave us the freedom to choose the all-trans, 6-s-trans conformation for retinal
(Harbison et al., 1985; van der Steen et al., 1986) with the plane of the chromophom nearly perpendicular to the membrane plane (Earnest et al:, 1986) and the methyl groups upwards (Lin & Mathies, 1989). The data presented by Lin & Mathies (1989) on the linear diehroism with vitamin A2, which has an extra double bond in the ionone ring, as chromophore gives an unambiguous indication of the methyl group direction when coupled with the definite determination of the vertical position of the retinal obtained from our density map. We themfore built the retinal as shown in Figures 14 and 16. The amino acid side-chains were built into the density as well as possible considering the relatively poor vertical resolution. Most of the Phe, Tyr and Trp side-chains in the bottom half of the helices are well-determined in position but not necessarily in orientation. The vertical resolution does not allow the orientation of side-chains to be determined,
V| 17 S6
')uI
Figurv 16. Stereo view of the retinal environment viewed from the cytoplasmic surface. The positions of residues Trp182 and Asp115 are slightly ambiguous. Nevertheless, both are very close to the ~-ionone ring.
R. Henderson et al.
920 Table
3
Helix parameters Helix A
B
Residues 10-32 38-62
C
80-101
D E F G
108-127 136-157 167-193 203-227
Number
Residue at same level as retinal ring
23 25 22 20 22 27 25
16 56 85 117 139 185 210
The residue number at which each helix terminates is uncertain at the top and bottom of each helix by at least 1 residue.
though sometimes the stereochemistry dictated the orientation. For example, the position of the sidechain of Tyr185 suggests that its ring plane must be parallel to the plane of the extended polyene of the retinal and therefore roughly vertical. Similarly, the rings of Trpl37 and Trp138 are likely to be vertical in orientation. The top parts of the density of most of the helices are weaker and suggest that the structure is intrinsically less well ordered so that positions of side-chains in the top one-third of the helices are less certain. Similarly, although the linking regions have reasonable density and a plausible model can be built, we cannot be so sure that interpretation of the links is not out by one residue in either direction. As mentioned above, the interpretations of the top half of helix F and all of helix D are also less certain. Nevertheless, we are confident of the interpretations shown in Figures 14, 15 and 16. Figure 14 shows two representations of the overall chain tracing. Figure 15 shows different views of the seven helices selected to show up the slight kinking at the three proline residues that are approximately in the centre of helices B, C and F. Helix G seems also to be slightly bent near Lys216. The other three helices are generally straighter. Figure 16 shows a stereo view of the retinal and its environment together with those side-chains that we are most certain about.
Retinal makes contact with Trp86, Trp138, Trpl82 and Trp189. Trpl37 is not in contact with the retinal. T y r l 8 5 and probably Tyr83 are in contact with the retinal, as are Thr89 and Thr90. Altogether, with a slight uncertainty because of the resolution and possible ambiguities, 21 amino acid residue~ in all (Table 4) form the retinal binding pocket. Having now described the retinal binding site, we turn to the residues that may be involved in forming the lower part of the proton channel. The position of Asp212 is such that Tyr57, Tyr185 and Trp86 are its closest neighbours, suggesting that the side-chain is stabilized by hydrogen bonds to the two tyrosine hydroxyl groups and the indolyl NH of Trp86. No density is resolved for Asp85, but the interpretation of the whole helix places Asp85 at a similar distance from the Schiff base as Asp212. Arg82 is below Asp85 and its side-chain could be placed either upwards near Asp85 or downwards away from Asp85, or could move between the two positions. Asp212 is stabilized by more interactions than Asp85. Overall, the environment below the Schiff base, seen in Figure 16, shows a fairly open structure with sufficient room for a reasonable number of water molecules and a constellation of very hydrophilic residues that would be accurately described as forming a hydrophilic and charge-rich channel between the Schiff base and the extracellular surface. In contrast, the region above the Schiff base looks like a narrow, close-packed and hydrophobic region with the single exception of the side-chain of Asp96, which is positioned l0 to 12 A above the Schiff base and 6 to 8 A from the end of helices G and C. Since it is known that Asp96 is the proton donor in the second half of the photochemical cycle in which the Schiff base is reprotonated, the region between Asp96 and the Schiff base must be the upper half of the proton pathway or channel. Thus the cytoplasmic half of the channel is narrow and hydrophobic, whereas the extracellular half of the channel is wider and hydrophilic. Residues lining the proton channel are shown in Figure 17, again with slight reservations because of the poor resolution. More
Table 4
Rexidues that line the retinal pocket Helix
A
Above
B
D
E
F
G
Val49 I~u93 Thrg0
retinal
Level with retinal
C
Met20
Below retinal
AlaS3
Trp182 Met145 Asp115
Thr89 Tyr57
Trp86 Asp85
Serl41 Trpl38
Tyrl85 Pro186
Lys216
Met118 Gly122
Trp189
Asp212
Note: 10 of these residuesat the Schiffbase end are in commonwith Figure 17 becausethey also line
the proton channel.
Model for Bacteriorhodopsin Structure
Cytoplasmic surface
Upper channel
Immediate retinal environment
Lower channel Extrocellular surface
Q
= Retinal binding pocket
Figure 17. Residues lining the proton channel.
precise information on the channel sizes and space available for water molecules should await refinement. Of 21 residues, listed in Table 4, close enough to the retinal to be in probable van der Waals' contact, 13 are identical in hR and 17 in sR. Overall, in the retinal binding site 62 ~/o of the residues are identical in all three proteins, whereas in the rest of the molecule the conservation over all three is only I0~/o . Again this gives us confidence that the interpretation is correct. In the proton channel (Figs 17 and 19), similar comparisons between bR, hR and sR are interesting. In the narrow upper channel, changes in hR make the size of the protein side-chains smaller by the equivalent of five methylene groups, whereas in sR the protein side-chains are larger by eight methylene groups. This suggests that the hR channel, for chloride ions, is larger, whereas in sR the channel must be narrower, and may be entirely blocked. 4. Discussion (a) High-resolution electron microscopy We have demonstrated that it is possible to record and analyse high-resolution images of highly tilted specimens of two-dimensional crystals. These images can then be used to determine the structure of, in this case, a membrane protein in atomic detail. The map we have obtained approaches a resolution typically obtained in X-ray diffraction analyses of protein crystals. More data are being
921
collected. This will give a map improved both in resolution, particularly in the vertical direction, and in noise level. The reason we are able to interpret the present map with some confidence is because the large amount of a-helical secondary structure makes interpretation easier, and because the large body of available structure-function correlations from mutagenesis confirms that the identifications of the features are all reasonable. Without these two valuable supports, we would need to continue to process more images before being able to interpret the density confidently. Although the best images we have obtained contain excellent contrast at all resolutions and in all directions, they are still far from attaining the best that is, in principle, possible. The best image of native purple membrane so far obtained is an image of an untilted specimen taken at liquid helium temperature in Berlin (Baldwin et al., 1988). I t shows an average peak-to-background ratio of diffracted intensity in the 3"5 to 5"5 A resolution range of 22 and significant power beyond that out to 2.8 A. As shown by Henderson & Glaeser (1985), even this is less than a good image should have with a perfect microscope and specimen. In the present work, using tilted specimens, the best peak-to-background ratio in the 3-5 to 5"5 A resolution range is 10 near the tilt axis, 6 perpendicular to the tilt axis for 20° tilted specimens and 2"5 perpendicular to the tilt axis in a 41 ° tilted specimen (see Fig. 5). The best image of a tilted specimen was taken at Berkeley using the spotscan procedure with highly coherent illumination (i.e. field emission gun). The remaining tasks that need to be tackled to make the use of image phasing a robust and reliable method in electron crystallography include a better understanding of specimen preparation; the behaviour of carbon support films is very variable. We need to understand why some specimens lose their crystallinity on some support films, and why some two-dimensional crystals can make flatter and smoother specimens than others. There is also the need to overcome the problem of specimen or image movement or blurring which, especially in tilted specimens, causes a great loss of high-resolution contrast. This is due either to physical movement of the specimen as it is damaged by the electron radiation or to build-up of charge during the exposure to electrons. In tilted specimens, the movement or blurring is often worse in the direction perpendicular to the carbon film, making good images of tilted specimens much more difficult to obtain. Clearly, spotscan (Henderson & Glaeser, 1985; Downing & Glaeser, 1986; Bullough & Henderson, 1987; Downing, 1988; Zemlin, 1989) provides a substantial improvement but at least one more technical advance would help. In any ease, it is clear that specimen cooling, preferably to liquid helium temperature, and the use of an illuminating beam that covers only a small area of the specimen at any instant (spotscan) both produce valuable improvements in image quality. For images of tilted specimens, in addition, the coherence associated with the
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electron beam from a field emission gun produces an envelope function that extends to high resolution even for regions of the image that are highly defocused. We conclude that no currently available microscope is perfect. The time is therefore ripe for a fresh effort to develop a microscope with all the features we now know are essential for this type of work. These are a stage temperature preferably near that of liquid helium, good positional stability, very high vacuum to avoid contamination of ice on the specimen, computer control of all microscope functions to allow spotscan imaging, a field emission gun for superb coherence, and ease of use in the normal operator environment provided by one of the major electron microscope manufacturers. A higher voltage than that presently available, say 200 or 400 kV would also be an advantage. This would allow the investigation of the remaining specimen problems that at the moment are difficult to untangle from the other problems that sometimes occur with the temperamental prototype cold stages and elderly microscopes that we have been using. Once there is a reliable and easy to use microscope, and the remaining problems of specimen movement and image blurring are overcome, it should be possible to record images regularly that are better than any yet obtained. This would then turn the technique we have been using into a routine and quick method, able to be used on many more difficult specimens, eventually including non-crystalline molecular assemblies. The future of high-resolution molecular structure determination by electron microscopy will then be even brighter than it is already. (b) Structure of bR: correlation with other work The vertical position of the retinal with the Schiff base in the centre of the protein and the ring about 5 A lower and nearer the extracellular surface gives a tilt angle of roughly 20 ° ( _ 10°) out of the membrane plane, in agreement with most other measurements, which range from 15° to 25 ° (Heyn et al., 1977; Earnest et al., 1986; Lin & Mathies, 1989). Previous attempts to determine the distance of the retinal ring from either membrane surface ~ have concluded that it is indeed nearer the extracellular surface by chemical cross-linking (Huang et al., 1982), by a second harmonic interference technique (Huang & Lewis, 1989), and by energy transfer (Leder, R. O., Helgerson, S. L. & Thomas, D. D., unpublished results). Otomo et al. (1988) have suggested that the ring is nearer the cytoplasmic surface, but Lin & Mathies (1989) and Leder et al. (unpublished results) explain the results presented by Otomo et al. (1988) as being due to higher energy transfer resulting from specific adsorption of their probes by the negatively charged cytoplasmic surface, thus resolving their disagreement. Huang et al. (1982) found that their ring-labelled retinal reacted with residues Ser193 and Glu194, whereas we find that the ring is some l0 A above Trp189~ level with
Pro186. However, if the retinal is allowed to swing downwards, pivoting about the Schlff base, then it can reach residues 193 and 194, which are ideally placed to react with a bulky ring-labelled retinal that is perhaps too big to fit into its normal binding site. Linear dichroism measurements before or after reaction to determfine the angle of the azidophenylretinal could test this explanation. Our position for the retinal ring, distant from residues 193 and 194 also explains why replacement of Gtu194 by Gin has no effect on the bR spectrum (Hackett et al., 1987). The neutron diffraction results of Hauss et al. (1990), which also indicate a non-central position for the retinal ring without specifying which side, are also in agreement. Lin & Mathies (1989) present the only measurement that determines in which direction the methyl groups point. We have assumed this is correct in building our model, since the resolution of our density map is too low to see the methyl groups directly. The position of the retinal viewed in a direction perpendicular to the membrane plane is in excellent agreement with recent neutron diffraction studies (Heyn et al., 1988). The polypeptide chain trace agrees with that previously deduced as being the most probable by Engelman et al. (1980), and subsequently used in nearly all efforts to present coherent accounts of a variety of data (Khorana, 1988; Oesterhelt & Tittor, 1989; Popot et al., 1989; Lin & Mathies, 1989). Of the arguments used by Engelman et al. (1980), the one that argued that the structure should contain the shortest links able to connect the ends of the low-resolution helix density can now be seen to be the most powerful, since the linking regions that contain longer stretches of polypeptide are now found to be physically compact, possibly forming short stretches of horizontal ~-helix. Similarly, the lower density found in the positions occupied by helices A and D was helpful. However, the suggestion that there would be a salt-bridge between Lys172 and Aspll5, and another between Arg175 and Asp96 is now seen to be wrong. The orientation of helix F does not allow it. With Tyr185 and Pro186 directed towards the centre, the side-chains of Lys172 and Arg175 are on the upper surface of the tilted helix, directed outwards from the centre of the molecule and to the right in Figure 14. Although they are not really high enough on helix F to be fully exposed to solvent, we presume they are able to reach the polar lipid headgroup region. This position for Arg175 seems consistent with the observation that the mutant R175Q has a tenfold slower folding rate (Stern & Khorana, 1989) and that Arg175 may have a role in correctly orienting helix F during folding. Neither the lipids nor the side-chains in this topmost part of the structure are sufficiently ordered to see them. The C terminus from residue 225 onwards is disordered, as shown previously (Wallace & Henderson, 1982). Agard & Stroud (1982) claimed to be able to reveal the link regions using a density modification procedure on much lower resolution data from Henderson & Unwin {1975). Some of the extra
Model for Bacteriorhodopsin Structure features that they found occur in positions where we now find density but others do not. For example, the link between the tops of helices C and D, which shows up as the clearest link in our map, was not observed, whereas the E F link, which was the only density at the top of their modified map, is in fact absent. Previous indications of link regions in the experimental maps (Leifer & Henderson, 1983; Tsygannik & Baldwin, 1987) are now confirmed at higher resolution in the same places as seen before. In their vertical positions across the membrane, the beginnings and ends of the helices (Table 3 and Fig. 19) are not substantially different from many previously proposed arrangements (e.g. Ovchinnikov et al., 1979), though perhaps helix D is physically positioned rather lower than in most proposals and helix F considerably earlier in the sequence and lower physically than in some (e.g. Engelman et aL, 1982; Dunn et aI., 1987). Nearly all the information from chemical labelling studies or from proteolytic enzyme and antibody accessibility studies, summarized by Ovchinnikov et al. (1985), is in good agreement. Within the plane of the membrane, neutron diffraction studies of specifically deuterated labels have shown the position and orientation of helices A and B (Popot et al., 1989), electron diffraction suggested a C terminus position (Wallace & Henderson, 1982), and neutron diffraction of deuterated, phenyl-isothiocyanate-labelled membranes suggested a position for Lys41 (Seiff et aI., 1986). Only the last of these studies is irreconcilable with the present model. Even an extended sidechain with a bulky phenyl group label cannot reach from the present position for Lys41 in the AB link to the peak in the difference map of the phenylisothiocyanate-labelled membranes. The retinal environment includes four tryptophan residues as found in energy transfer experiments (Polland et al., 1986). Three of these, Trp182, Trp189 and Trp86, were identified by Mogi et al., (1989b) on the basis of results from site-directed mutagenesis. Two detailed models for the environment of the retinal have been proposed, based on these and other results (Rothschild et al., 1989a; Lin & Mathies, 1989). Both propose that Trp182 and Trp189 sandwich the central polyene of the retinal, as also suggested by Oesterhelt & Tittor (1989). In addition, Rothschild et al., (19895) include Trp86 as part of the proposed retinal pocket. The position that we find for Trp182 is indeed above the centre of the retinal, whereas Trp189 is below the fi-ionone ring. The sandwich is formed by Trp182 above and both Trp86 and Trp189 below the retinal. The positions that we find for the residues of helix F are certain, since they are determined by the position of the retinal ring, the position and tilt of helix F and the interpretation of the resolved densities for the bulky aromatic side-chains. In the model proposed by Rothschild et al. (1989a) and Braiman et al. (1988a,b) based on vibrational spectroscopy of sitedirected mutants, Tyr185 is placed close enough to the Schiff base to interact directly and they propose that the tyrosine hydroxyl is deprotonated and
923
forms a counterion at the beginning of the photocycle. In our structure, the tyrosine hydroxyl group is 6 A from the Schiff base, so this is not possible without considerable and unlikely helix movements during the photocyele. Instead, Tyr185 seems to form a hydrogen bond to the negatively charged Asp212 side-chain, producing an indirect interaction with the Schiff base. The fiat ring of Pro186 is in contact with the fl-ionone ring of the retinal, and could explain the observed effects of replacement of Pro186 by giycine, alanine, valine and leucine residues (Mogi et al~, 1989a). ~I~he arrangement proposed by Lin & Mathies (1989) also comes close to our findings, although the rotation of their helix F by 90 ° removes the close contact between Tyr185 and the extended polyene of the retinal that we find. In summary, the previously proposed models have turned out to be remarkably good, especially where they have been guided most closely by data. (c) Proton pumping In Figure 18, we show a diagram indicating the locations of Asp96, Asp85, Asp212 and Arg82, which line the proton channel, and their relation to the retinal ancl Lys216. These residues are the most important in the light-driven proton pumping. Tyr57 and Tyr185 are also close to the active site and both seem to make hydrogen bonds with Asp212. However, Mogi et al. (1987) have shown that the mutants Y57F and Y185F are still substantially active as proton pumps, whereas mutations to Asp85 or Asp96 or, to a lesser extent, Asp212, inhibit proton pumping (Mogi et al., 1988). Stern & Khorana (1989) have further shown that Arg82 affects the pK of one or both of the aspartic acid residues but, apart from this general effect, does not affect proton pumping. A minimal model for proton pumping would therefore involve the three aspartic acid residues and the Schiff base. The role of Asp96 has been established recently as that of the proton donor in the latter part of the photochemical cycle when the Schiff base, deprotonated at M412 is reprotonated to form N56o (Butt et al., 1989; Gerwert et al., .1989; Tittor et al., 1989; Holz et al., 1989; Otto et al., 1989). Its position 10 to 12 A above the Schiff base and the fact that it is protonated prior to M (Braiman et al., 1988a; Gerwert et al., 1989) is entirely consistent with this function. We assume that there must be at least one or two structured water molecules in the otherwise very hydrophobic channel above the Schiff base. The side-chains of Asp85 and Asp212 are approximately equidistant from and 4 A below the Schiff base and, since they are both unprotonated in bRs7 o, could be considered equally good Candidates for being counterions to the Schiff base in bRsTo (Braiman et al., 1988a; Gerwert et a/., 1989). However, on release of the Schiff base proton to form M412, we presume that only one of the two carboxyl groups acts as a proton acceptor. On the basis of the more hydrophilic surroundings of Asp212, we think that Asp85 is the more likely group to a c t as the
924
R. Henderson et al.
Figure 18. Artistic impression showing the relationship between the key residues Asp85, Asp96, Asp212, Lys216 and Arg82, and the retinal binding site, the proton channel and overall molecular boundary for the ground state of bR (bR57o). The cytoplasm is at the top of the diagram.
proton acceptor, consistent with the results of Mogi et al. (1988) that the mutation of Asp85 to asparagine has a much larger effect on proton pumping than the same change to Asp212. The role of Asp85 as proton acceptor in the transfer from the Schiff base to the extracellular surface is supported by the interpretation of Fourier transform infra-red measurements, which suggest that Asp85 is unprotonated in bR, K and L and protonates in M (Braiman et al., 1988a). It is supported by the fact that the D85E mutation affects the rate of formation of M (Butt et al., 1989) but retains some pumping activity, whereas D85N is completely inactive. Finally, Asp212 is unchanged whilst Asp85 is changed to Thr in hR, which pumps chloride instead of H + andhas no deprotonated M intermediat6: We therefore favour Asp85 as the site to which the proton is transferred from the Schiff base in spite of the fact that Braiman et al. (1988a) and Gerwert et al. (1989) interpret their Fourier transform infra-red measurements to indicate that both Asp85 and Asp212 become protonated in M. It should also be noted that this open arrangement on the extraceltular side of the Schiff base is consistent with the rapid proton exchange at the Schiff base observed by nuclear magnetic resonance spectroscopy and other methods (Harbison et al., 1988~. Similarly, the collection of charged residues in this region is consistent with the idea of a complex counterion for the protonated Schiff base that has.been inferred from nuclear magnetic resonance d a t a (De Groot et al., 1989).
In a minimal mechanism of proton pumping, shown schematically in Figure 20, we assume that the isomeric state of the retinal as derived from interpretations of resonance Raman data is correct (Smith et al., 1983, 1984, 1986; Fodor et al., 1988). It is necessary to propose transient protonation changes only to Asp96 and Asp85, leaving Asp212 permanently deprotonated and Aspll5, situated near the fl-ionone ring, permanently protonated. On isomerization of the retinal, the Schiff base pK becomes lower when it is connected via Asp85 to the extracellular surface, and subsequently returns to a high p K when it is connected v/a Asp96 to the cytoplasmic surface. The corresponding proton movements result in pumping. Clearly, Asp212 and A s p l l 5 would still be necessary in providing the correct charge environment at both the Schiff base end and the fl-ionone ring end of the chromophore. Structural changes in the different intermediates, which probably involve changes in Asp212 and Aspll5, must affect both the absorption spectrum of the retinal and the effective pK of the Schiff base. The effective p K values of Asp85 and Asp96 may also change but this is not essential to the discussion. In the minimal mechanism, the all-trans to 13-cis isomerization at the K intermediate first moves the protonated nitrogen of the Schiff base to a new environment more distant from the side-chain of either Asp85 or Asp212 or both. A conformational change in the protein, which could be quite small (e.g. it might include rotation of the carboxyl group
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925
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Figure 19. Our alignment of sR, bR and hR sequences with the helical segments outlined by a box and the correct relative heights indicated. The height of the retinal ring is at the level of Tyr185 and the cytoplasm is at the top. It can be seen that a very large proportion of the conserved residues line the retinal binding pocket (diamonds) but there is less conservation of residues in the proton channel (circles).
of Asp85 about its =-fl bond), to form L brings the Schiff base closer to Asp85 and into an environment to favour proton transfer in the formation of M. In the transition from L to M, a proton is released at the extracellular surface (Lozier et al., 1976), so the effective pK of both the Schiff base and Asp85 must be low. In order that the protonated Asp96, which has a high pK, can then transfer a proton to the Schiff base to form N, a second structural change in the protein must follow before the transition from late M to N so that the p K values and accessibility between Asp96 and the unprotonated Schiff base create conditions that favour the second proton transfer. Here, we call early M the structure formed by proton transfer from the Schiff base in L. This discriminates between the low p K state after proton transfer and a later high pK M state, which is required before reprotonation can occur. This late M intermediate is the form that would be observed kinetically, whereas early M is likely to be a shortlived, kinetically non-observable and therefore hypothetical intermediate. Finally, Asp96 must be reprotonated and the retinal must re-isomerize to form O~40 and convert back to hR. At least three
distinct protein structures are involved, best characterized by the K to L transition, the early M to late M transition and the 0 to b R return to ground state though, in practice, it might be more complex. For example, there is beginning to be evidence (Otto et al., 1989) that the reprotonation of Asp96 is necessary before the re-isomerization of the retinal to form 0. This would imply a further change of structure between N and O. At any of these stages, but it would be expected particularly in L and early M, the retinal may be in a strained out-of-planar conformation, which would certainly lower the Schiff base p K (Schulten & Tavan, 1978; Fahmy et al., 1989). Apart from distortions to make the retinal non-planar, two other effects seem likely to play a role in lowering the p K of the Schiff base in L and early M. The environment of the Schiff base may be more hydrophobic than it is in bR, N and O, and the side-chain of A s p l l 5 may be placed further from the ring of the retinal. ~ this discussion, protein structure changes play an important role in the mechanism, as they do in the C-T switch proposal of Fodor et aL (1988}, though the points at which changes might occur are
926
R. Henderson et al.
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2121
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(b)
Figure 20. A diagram to describe the 4 proton movements in the photocycle of bR. The only observed structure is bR. The others with the 13-cis isomerization of the retinal are speculations in which the isomeric state of the retinal is taken directly from interpretations of resonance Raman data (Smith et al., 1983, 1984, 1986; Fodor et al., 1988). It is proposed that the protein takes up at least 3 distinct conformations; (1) as in bR and K; (2) as in L and early M to facilitate the proton transfer to Asp85 to form M; (3) as in late M~and N to facilitate the proton transfer from Asp96 to form N. The Schiff base pK must be low in L and early M, and high in bR, late M, N and O. However, the precise nature of the interactions with the protein and the possible extent of out-of-plane distortions of the retinal that together determine the Schiff base pK and its position with respect to Asp85 in the lower proton channel and Asp96 in the upper proton channel are not yet obvious. (a) Structure of bR before isomerization of retinal. (b) Proton transfer from Sehiff base to Asp85. (c) Proton transfers from Asp96 to Schiff base and from Asp85 to the extracellular space. The timing of the transfer from Asp85 to the extracellular space is not yet clear. (d) Proton transfer from cytoplasmic surface to Asp96. different. Figure 20 shows only a minimal mechanism. The roles of Arg82, Tyr57 and Tyr185, as well as other serine and threonine residues and w a t e r molecules, have been omitted from the discussion, since they are not necessary to understand the basic mechanism. W a t e r molecules in particular, whether in the narrow, hydrophobic channel above the Schiff base or in the wider, hydrophilic channel below the Schiff base, are certain to play a role in
proton transfer as discussed by Nagle & Morowitz (1978}. Gating of the p a t h for the proton from the Schiff base first to the extracellular side, then from the cytoplasmic side might be mediated b y appropriate changes in hydrogen bonds without any change in the channel structure. Alternatively, it is possible t h a t the structural changes to form N with Asp96 u n p r o t o n a t e d cause the narrow hydrophobic chan-
Modal for Bacteriorhodopsin Structure nel above the Schiff base to open slightly so that solvation of the Asp96 carboxylate group is improved. In either case, it seems likely that the structural changes are relatively small. A number of measurements of charge movement across the membrane during the different steps of the photocycle have been made (Keszthelyi & Ormos, 1980; Trissl & G/~rtner, 1987; Butt et al., 1989; Otto et al., 1990). In most recent analyses, about 80 ~o of the charge movement occurs during the last part of the photocycle as M returns to bR (Butt et al., 1989). This is consistent with our finding that the upper proton channel is narrow and hydrophobic and is therefore the principal electrical barrier to movement of the proton across the membrane. Although the lower, extracellular proton channel is of equal length, its wider, hydrophilic character means it has space for water molecules and would present a lower barrier to movement of the proton. We expect that a more detailed understanding of the structure and mechanism of bR will be helped by higher-resolution analysis and refinement of the structure using the present model as a starting point. Further comprehensive analysis of the many different mutants is also critical with the eventual goal of a detailed dissection of the energetics of proton pumping.
(d) Refinement of the structure and study of structural changes Given a reasonable starting atomic model, such as in the present case, and diffraction amplitudes that extend beyond 3/k and up to 60 ° tilt (Ceska & Henderson, 1990), it should be possible to carry out a constrained refinement of the structure (Konnert & Hendrickson, 1980). This should give a more accurate structure, particularly in the vertical direction. Such refinement is in progress. I t should also be possible to investigate the structure of mutants, by the calculation of difference Fourier maps, if the mutants make good quality two-dimensional crystals. Glaeser et al. (1986) have demonstrated t h a t it is possible to trap an intermediate, which they believed to be M, at low temperature and collect good electron diffraction data. To get the greatest benefit from difference Fouriers, the errors in the diffraction data obtained must be less than the change due to the alteration of structure. In the case of bacteriorhodopsin electron diffraction data, we find the spread in amplitude measurements (overall mean value A A / A ) for typical 3 A diffraction patterns is about 8 to 1 0 ~ , suggesting that some improvements in accuracy are still required before difference Fouriers can show up some of the small'Changes that are expected to occur in the different photochemical intermediates. This is also a challenge for the future. The preliminary co-ordinates used in this model of bR are available on request from the authors and has been submitted to the Brookhaven data bank
927
together with the 2700 structure factors used to calculate the map. We thank EMBO for fellowships to R.H. and F.Z. and JEOL for supporting a visit by R.H. to use their 2000 SCM. K.H.D. was supported by grant number GM36884 from the US National Institutes of Health. We thank Jane Austin, Claudio Villa and staff of the photographic department at Cambridge for their help in producing the manuscript and illustrations. Finally, we thank our colleagues for their valuable comments on the manuscript and for their support and encouragement throughout this work. References Agard, D. A. (1983). J. Mol. Biol. 167, 849-852. Agard, D. A. & Stroud, R. M. (1982). Biophys. J. 37, 589-602. Argos, P., Rao, J. K. M. & Hargrave, P.A. (1982). Eur. J. Biochem. 128, 565-575. Baldwin, J. & Henderson, R. (1984). Ultramicroscopy, 14, 319-336. Baldwin, J. M., Henderson, R., Beckman, E. & Zemlin, F. (1988). J. Mol. Biol. 202, 585-591. Blanck, A. & Oesterhelt, D. (1987). EMBO J. 6, 265-273. Blanck, A., Oesterhelt, D., Ferrando, E., Schegk, E. S. & Lottspeich, F. (1989). EMBO J. 8, 3963-3971. Braiman, M. S., Mogi, T., Marti, T., Stern, L.J., Khorana, H. G. & Rothschild, K. J. (1988a). Biochemistry, 27, 8516-8520. Braiman, M. S., Mogi, T., Stern, L. J., Hackett, N. R., Chao, B. H., Khorana, H. G. & Rothschild, K. J. (1988b). Proteins: Struct. Funct. Genet. 3, 219-229. Bullough, P. & Henderson, R. (1987). Ultramicroscopy, 21, 223-230. Butt, H. J., Fendler, K., Bamburg, E., Tittor, J. & Oesterhelt, D. (1989). EMBO J. 8, 1657-1663. Ceska, T. A. & Henderson, R. (1990). J. Mol. Biol. In the press. Crepeau, R. H. & Fram, E. K. (i981). Ultramicrosco'py, 6, 7-18. Crowther, R. A. & Sleytr, U. B. (1977). J. Uitrastruct. Res. 58, 41-49. De Groot, H. J. M., Harbison, G. S., Herzfeld, J. & Griffin, R. G. (1989). Biochemistry, 28, 3346-3353. Dietrich, I., Fox, F., Knapek, E., Lefranc, G., Nachtrieb, K., Weyl, R. & Zerbst, H. (1977). Ultramicroscopy, 2, 241-249. Downing, K. H. (1988). Ultramicroscopy, 24, 387-398. Downing, K. H. & Glaeser, R. M. (1986). Ultramicroscopy, 20, 269-278. Dunn, R., McCoy, J., Simsek, M., Majumdar, A., Chang, S. H., Ra]Bhandary, U. L. & Khorana, H. G. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 6744-6748. Durra, R. J., Haekett, N. R., McCoy, J. M., Chao, B. H., Kimura, K. & Khorana, H. G. (1987). J. Biol. Chem. 262, 9246-9254. Earnest, T. N., Roepe, P., Braiman, M. S., Gillespie, J. & Rothschild, K. J. (1986). Biochemistry, 25, 7793-7798. Eisenstein, L., Lin, S.-L., Dollinger, G., Odashima, K., Termini, J., Konno, K., Ding, W.-D. & Nakanishi, K. (1987). J. Amer. Chem. Soc. 109, 6860-6862. Engelhard, M., Gerwert, K., Hess, B., Kreutz, W. & Siebert, F. (1985). Biochemistry, 24, 400-407. Engelman, D. M. & Zaccai, G. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 5894-5898.
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Model for Bacteriorhodopsin Structure Saxton, W. O. & Baumeister, W. (1982). J. Microsc. 127, 127-138. Schiske, P. (1982). Ultramicroscopy, 9, 17-26. Schulten, K. & Tavan, P. (1978). Nature (London), 272, 85-86. Seiff, F., Wallat, I., Ermann, P. & Heyn, M. P. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 3227-3231. Seiff, F., Wallat, I., Westerhausen, J. & Heyn, M. P. (1986}. Biophys. J. 50, 629-635. Shaw, P. J. & Hills, G. J. (1981). Micron, 12, 279-282. Smith, S. 0., Pardoen, J. A., Mulder, P. P. J., Curry, B., Lugtenburg, J. & Mathies, R. (1983). Biochemistry, 22, 6141-6148. Smith, S. 0., Myers, A.. B., Pardoen, J. A., Winkel, C., Mulder, P . P . J . , Lugtenberg, J. & Mathies, R. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 2055-2059. Smith, S. 0., Hornung, I., van der Steen, R., Pardoen, J. A., Braiman, M. S., Lugtenburg, J. & Mathies, R.A. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 967-971. Soppa, J. & Oesterhelt, D. (1989). J. Biol. Chem. 264, 13043-13048. Soppa, J., Otomo, J., Straub, J., Tittor, J., Meessen, S. & Oesterhelt, D. (1989). J. Biol. Chem. 264, 13049-13056.
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Stern, L. J. & Khorana, H. G. (1989). J. Biol. Chem. 264, 14202-14208. Tittor, J., Soell, C., Oesterhelt, D., Butt, H.-J. & Bamburg, E. (1989). EMBO J. 8, 3477-3482. Trewhella, J., Popot, J. L., Zaccai, G. & Engelman, D. M. (1986). EMBO J. 5, 3045-3049. Trissl, H.-W. & G~/rtner, W. (1987). Biochemistry, 26, 751-758. Tsuda, M., Glaccum, M., Nelson, B. & Ebrey, T. G. (1980). Nature (London), 287, 351-353. Tsygannik, I. N. & Baldwin, J. M. (1987). Eur. J. Biophys. 14, 263-272. Unwin, P. N. T. & Henderson, R. (1975). J. Mol. Biol. 94, 425-440. van der Steen, R., Biesheuvel, P. L., Mathies, R. A. & Lugtenburg, J. (1986). J. Amer. Chem. Soc. 108, 6410--6411. van Heel, M. & Hollenberg, J. (1980). In Electron Microscopy at Molecular Dimensions (Baumeister, W. & Vogell, W., eds), pp. 256--260, Springer, Berlin. Wallace, B. A. & Henderson, R. (1982). Biophys. J. 39, 233-239. Zemlin, F. (1989). J. El. Microsc. Tech. 11,251-257. Zemlin, F., Weiss, K., Schiske, P., Kunath, W. & Hermann, K.-H. (1978). Ultramicroscopy, 3, 49-60.
Edited by D. DeRosier
Model for the Structure of Bacteriorhodopsin Based on High-resolution Electron Cryo-microscopy R. H e n d e r s o n , J. M . Baldwin, T. A. C e s k a t
MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH, U.K. F. Zemlin, E. B e c k m a n n
Fritz-Haber-Institut der Max-Planck-Gesellschafl Faradayweg 4-6, D-IO00 Berlin 33, F.R.G. and K. H. D o w n i n g
Cell and Molecular Biology Division Lawrence Berkeley Laboratory Berkeley, CA 94720, U.S.A. (Received 20 December 1989; accepted 31 January 1990) The light-driven proton pump-bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full threedimensional transform to 3"5 A. The map of the structure has a resolution of 3"5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven ~-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the fl-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.
tPresent address: EMBL, Heidelberg, F.R.G.
0022-2836/90/120899-31 $03.00/0
899
~) 1990AcademicPressLimited
R : Henderson et al.
900 1. Introduction
Bacteriorhodopsin (bRt) is a membrane protein that functions as a light-driven proton pump in Halobacterium halobium (Oesterhelt & Stoeckenius, 1973). The protein forms a 1 : 1 complex with the retinal chromophore, which gives the protein its characteristic purple colour (Oesterhelt & Stoeckenius, 1971). The polypeptide takes up a conformation consisting of seven transmembrane ahelices (Henderson & Unwin, 1975) with the retinal in the middle (Jubb et al., 1984; Seiff et al. 1985). A concise review of our present understanding of the structure of bR, and the role of many of the amino acids in the proton pumping mechanism has been given by Khorana (1988). Briefly, the structure is known at low resolution from the analyses of three independent crystal structures, which have been averaged (Tsygannik & Baldwin, 1987). The amino acid sequence (Ovchinnikov et al., 1979; Khorana et al., 1979), confirmed from the DNA sequence (Dunn et al., 1981), led to a proposal for the path of the polypeptide (Engelman et al., 1980), which also agrees with more recent data (Khorana, 1988). The sequence can be found later, in Figure 19. The orientation of the cytoplasmic surface in the crystal is known from labelling studies and freeze-fracture (Henderson et al., 1978; Hayward et al., 1978), and the retinal position in projection is known from neutron diffraction (Jubb et al., 1984; Seiff et al., 1985; Heyn et al., 1988). In addition, neutron diffraction from specifically deuterated specimens has led to the unequivocal determination of the positions of several of the helices (Engelman & Zaccai, 1980; Trewhella et al., 1986; Popot et al., 1989), again in agreement with the polypeptide connectivity proposed by Engelman et al. (1980). Khorana and his colleagues have investigated a large number of single amino acid mutants of bR, which they have engineered in an Escherichia coli expression system (Khorana, 1988). Specifically, they have shown (Mogi et al., 1988) that mutations to Asp85, Asp96 and, to a lesser extent, Asp212 have profound effects on proton pumping. They have shown (Mogi et al., 1987} that Tyr57 affects the kinetics of chromophore regeneration from free retinal and opsin, and that Tyrl85 has a smail but clear effect on pumping. Just as important, they have shown that changes in any of the other tyrosine or aspartic acid residues do not affect pumping. The absorption spectrum is perturbed by changes to many more amino acid residues including Trp86, Trp137, Trp182 and Trp189 as well as Arg82, Thr89 and Aspll5. Many of these may form the retinal binding site (Khorana, 1988). Mutants defective in proton pumping have been obtained by mutagenesis of bR in Halobacterium strain GRB (Soppa & Oesterhelt, 1989), including changes of Tyr57, tAbbreviations used: bR, baeteriorhodopsin; hR, halorhodopsin; sR, sensory rhodopsin; r.m.s., root-mean-square.
Asp85, Asp96, Trp138 and Trpl0 (Soppa et al., 1989). Such data provide strong evidence to indicate the orientations of many of the a-helices in the structure, showing which "side-chains must point inwards towards the retinal binding site and the pathway or channel through which the proton must move. In general, orientations derived from mutagenesis are in agreement with the idea that the helices should be oriented with their hydrophobic faces pointing outwards towards the lipid and their hydrophilic faces pointing inwards to the active site (Engelman & Zaccai, 1980; Argos et al., 1982). Two other retinal proteins from halobacteria, together with bR, make up a family of homologous membrane proteins. They are the light-driven chloride ion pump, halorhodopsin (hR) and the phototactic pigment, sensory rhodopsin (sR). The protein sequences of both of these proteins have been determined from the DNA sequences, hR shows 32% identity to bR (Blanck & Oesterhelt, 1987), and sR 26~/o (Blanck et al., 1989). Many (14~/o) of the residues are identical in all three proteins, including most of those known to be functionally important from the mutagenesis of bR. In addition, there is a clear indication of a greater degree of homology among amino acids that are expected to be directed inwards towards the common retinal binding site, giving further evidence that the binding site is one of the most tightly conserved characteristics of the family. A concise review pointing out the similarities between bR and hR has been written by Oesterhelt & Tittor (1989). Linear dichroism measurements have shown that the retinal absorption dipole is oriented at an angle of about 20° to the membrane plane (Heyn et al., 1977). Attempts to determine the retinal position normal to the membrane by fluorescence energy transfer, neutron diffraction and other methods have given contradictory results. However, the formation of a Schiff base b~tween the retinal and the e-amino group of Lys216, which is roughly in the middle of the hydrophobic stretch of amino acid residues that marks the C-terminal helix, means that the Schiff base of the retinal must be located approximately halfway between the cytoplasmic and extracellular surfaces of the protein. Extensive kinetic and spectroscopic analyses (Lozier et al., 1975; Kouyama et al., 1988) have resulted in the nearly universal adoption of the scheme for the photocycle shown in Figure 1. The main steps in the principal photocycle are indicated, starting from the light-adapted ground state bRs~o, which has retinal in the all-tran8 conformation. Much is known about the conformation of the retinal and protonation state of the Schiff base in each of the photochemical intermediates, from resonance Raman spectroscopy (Smith et al., 1983, 1984, 1986; Fodor et al., 1988), 13C nuclear magnetic resonance studies (Harbison et al., 1985), and analysis of the isomeric state of extraction products (Pettei et al., 1977; Tsuda et al., 1980). Fourier transform infra-red difference spectroscopy between the different intermediates has
Model for Bacteriorhodopsin Structure
/
bR570
40 "¢ t~
N560
K610 /
L550
901
perpendicular to the membrane, can be considerably improved b u t we present these preliminary results now because they provide the first near-atomic resolution picture of b R in which chemically distinct groups of the protein can be discerned. We are able to present a model for the protein t h a t includes clear positions for m a n y of the most i m p o r t a n t sidechains, including nearly all the residues t h a t form the retinal binding site and those lining the p r o t o n channel.
2. M e t h o & o e
po"H
Figure 1. A scheme for the photochemical cycle of bR based on that proposed by Lozier et al. (1975). The cycle is initiated by light absorption by bRs70. A proton is released on the outside and another taken up from the inside at the steps indicated.
shown changes in aspartic acid side-chains (Engelhard et al., 1985; Eisenstein et al., 1987) and, to a lesser extent, in a tyrosine side-chain (Rothschild et al., 1986). When applied to the series of aspartic acid and tyrosine mutants, the results have suggested which of the i m p o r t a n t active site residues are p r o t o n a t e d and which are deprotonated in each of the photochemical intermediates (Braiman et al., 1988a,b; Gerwert et al., 1989). Taken together, all these d a t a form a formidable body of knowledge and have encouraged Rothschild et al. (1989a), F o d o r et al. (1988) and Lin & Mathies (1989) to suggest models for the retinal binding site and to propose mechanisms for proton pumping. However, there is still a need for a knowledge of the three-dimensional structure obtained by a direct structural method. I t is essential to prove which parts of the structure are directly involved in proton pumping and which only indirectly, either v/a more distant interactions or through their role in providing a structural framework for the groups t h a t are directly involved. We report here the results of an electron microscopic analysis, based on methods first used by Unwin & Henderson (1975), of the structure of b R in the naturally occurring purple membrane in a state close to t h a t found in the halobacterial membrane. The map represents the highest resolution so far obtained in the three-dimensional analysis of protein structure using electron crystallography to measure the amplitudes and phases of the Fourier components of the unstained structure. Although several two-dimensional crystal forms of b R are available, we have chosen to work initially on the native crystal form (space group/93, with a = 62.45 A: 1 A = 0.1 nm) because it forms the most stable andTeproducible crystals. The resolution we have reached, 3"5 ~ parallel and 10 A
Specimen preparation Purple membranes from H. halobium, prepared by the (a)
method of Oesterhelt & Stoeckenius {1974), were fused to make much larger 2-dimensional crystals using the method of Baldwin & Henderson (1984). Briefly, a suspension of 3 mg purple membrane]ml (100/tM-bR) in 0"l M-potassium phosphate (pH 5"2}, containing 6mM-octyl glucoside and 200#M-dodecyl trimethyl ammonium chloride is incubated for several weeks at 20°C. These fused membranous sheets consist of a single layer of bR molecules, which anneal into large areas of homogeneous single crystals. Because about half of the bR molecules in each fused sheet are upside down with respect to the other half, the fused membranes contain a patchwork of crystalline areas that are related to one another by twinning (2-fold axes) perpendicular to and parallel to the membrane plane. The largest untwinned areas are normally 5 to 10/~m in diameter, whereas the largest sheets are about 20 #m in diameter (Baldwin & Henderson, 1984). Since the size of the untwinned areas was generally larger than that required (5 to 7 #m) to record a cryo-eleetron diffraction pattern from a 60° tilted specimen and much larger than that required (0"8~m) to cover the field of view of an image, most of the electron diffraction patterns were from untwinned crystals, whereas none of the images so far analysed has shown twinning. For the electron microscopy, support films were prepared by evaporation of carbon on to a freshly cleaved mica surface, floating off the film on water and then lowering it gently on to 400-mesh or 700-mesh copper grids. The grids were aged in air for 4 to 8 days at room temperature before use. Specimens were then prepared by application of a drop of the fused membrane suspension to the carbon film for 30 s, removal of most of the drop by touching the edge of the grid against filter-paper, addition of a drop of 0-8% (w/v) glucose solution (Unwin & Henderson, 1975), followed by final removal of the entire drop using a second piece of filter-paper. The procedure is the same as that used by Baldwin & Henderson (1984), but some further observations might be useful. When the carbon film is fresh (less than 1 day old), it tends to be very hydrophilic. Consequently, many membranes stick to the film and the distribution looks very good; it is easy to find many large flat membranes. However, the crystallinity is usually impaired by a disordering of the lattice, which shows up as a weakening and blurring of the highresolution reflections. As the carbon film ages, it becomes more hydrophobie, and fewer membranes stick to it, but many more diffract strongly, sharply and without fall-off, presumably because there are fewer distortions through contacts with the substrate. Once the carbon film is very old (1 to 2 months), almost no membranes stick and all are washed away when the final drop of glucose is
R. Henderson et al.
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removed by blotting. Also, on very old, hydrophobic grids the diffraction patterns from occasional membranes that do stick are again poorer. Thus, there is an optimum in the age of the carbon film at about a week. Since the fusion mixture contains detergent, the initial drop applied to the grid always spreads out and wets the carbon surface. By varying the volume of fusion mixture allowed to remain as a thin film on the grid before application of the glucose wash, it is possible to vary the composition and behaviour of the film of liquid that dries to form the final embedding medium. It is thus possible to use carbon films with differing behaviour to make excellent specimens. Basically, if the carbon film is more hydrophilic, then less fusion mixture is allowed to remain (and therefore less detergent), whereas if the grid is older and more hydrophobic, more fusion mixture is allowed to remain. In this way, good specimens (i.e. those with several membranes on each grid square, all diffracting well) could be made with a range of carbon films. Sometimes, the mica side of the carbon film gave better results than the air side. Also, ff the specimens had been prepared by leaving a larger proportion of the detergent in the final dried film (the ratio of octyi glucoside to glucose was as high as 1:15), the resulting grids did not last long. :Frequently, all diffraction would be weak or absent 24 h later. Thus, it was necessary to store the prepared grids in grid boxes in solid CO2 at about -80°C, particularly if they had been prepared with a substantial level of residual detergent. This also proved to be a convenient way of transporting the grids reliably between the different laboratories. (b) Electron diffraction A description of the electron diffraction methods is given by Baldwin & Henderson (1984). A complete set of electron diffraction data has been collected at -120°C using a Philips cold holder (PW 6599/00) on a Philips EM420 instrument, operated at 120 kV. The data scaling and merging is described by Ceska & Henderson (1990). Briefly, 150 electron diffraction patterns taken almost entirely in the form of patterns from 60° tilted specimens were scaled, merged and detwinned where necessary, to give a set of 2-9 A amplitudes complete apart from the missing cone. The coefficients describing the continuous diffraction amplitudes along the lattice lines (at intervals of 0"01 A -1) have been used as input data in the final curve-fitting using amplitudes and phases together. (c) Electron microscopy During this work, the technical side of recording the images has been most demanding and remains, with specimen preparation, as the most critical part of the work. To make progress and try to discover why the images are so much less perfect than they should be (Henderson & Glaeser, 1985), we have collaborated in using several microscopes, each of which has some advantages and some disadvantages. Of the 72 images used to determine phases, most of them (52) were recorded in Berlin on a prototype Siemens microscope, the Suleika, operating at 100 kV and liquid helium temperature (Dietrich et al., 1977). A smaller number of images (11) were included from images recorded at Berkeley on a JEM-100B operating at 100 kV and -115°C and equipped with a field emission gun. The Berkeley images were all recorded using the spotsean procedure (Henderson & Glaeser, 1985; Downing & Glaeser, 1986; Bullough & Henderson, 1987; Downing, 1988; Zemlin, 1989). Spotscan images are
recorded with a 500 to 1000 A diameter illuminating beam, which is scanned stepwise across the area to be imaged on the film. This results in a greatly decreased amount of beam-induced movement of the specimen and therefore much less blurring of the image. In spite of the much higher temperature, the best of the images from tilted specimens taken in Berkeley with spotscan were better in the tilted direction than those taken without spotscan in Berlin. The use of spotscan and the coherence of the field emission gun are clearly important for this improvement, especially for tilted images. However, by optical diffraction, the images taken at -115°C tended to have fuzzier diffraction spots than those at liquid helium temperature, which were always extremely sharp. Finally, a few images (6) were also recorded in Cambridge at liquid nitrogen temperature using a home-made side-entry cold holder (Henderson et al., unpublished results), and 2 test images were taken in Japan using the prototype Jeol 2000 SCM operating at 160 kV, with the same lens design as the Suleika. Details of the conditions used in the Berlin and Berkeley microscopes are given below. In each case, the images used in the subsequent computer analysis were carefully selected by optical diffraction, as described in Results, from a much larger number actually recorded, probably from several thousand images if we include those taken during the efforts to improve the success rate. In a good recording session (using spotscan method with an excellent grid), about half of the images obtained were good enough to include in the data processing, whilst in a bad session several hundred images might be recorded without one being good enough. (i) Liquid helium temperature in Berlin The helium-cooled superconducting cryo-electron microscope in Berlin, called SULEIKA (Supraleitender Kryoapparat) was designed by I. Dietrich and co-workers at Siemens, Munich (Dietrich et al., 1977; Lefranc et al., 1982). The main advantage of this microscope is the very low specimen temperature of -268°C, resulting in reduced radiation damage. We found by experience that a dose of 20 e/A 2 is optimal for imaging. This is perhaps l0 times greater than the optimal dose in an image of a room-temperature specimen. Some other advantages of the Suleika objective should be mentioned. (1) The magnetic field, produced by the superconducting coil and formed by the superconducting shielding, is extremely constant, like that of a permanent magnet. (2) The large pole-piece gap of 7"5 mm allows large specimen tilt. (3) The vacuum in the environment of the specimen is better than 10 -~ Torr (1 Torr ~ 133 Pa) and free of water and hydrocarbons, because the specimen is in the middle of a powerful helium-cooled "cryopump", so there is no measurable ice deposition on the specimen. The imaging procedure consists of 3 steps. In step 1, we search for a crystal. The electron optics is set to a "defocused diffraction" mode with a magnification of 500 x and a dose rate of 0.01 e A -2 s -l. The specimen is scanned by observing the image via a television camera and monitor. About 10s (0"1 e/A 2) is needed to decide whether a crystal is suitable for imaging. From time to time the crystallinity of the specimen is checked by diffraction. This requires a further 1-0 e/A 2. Before making the adjustments needed for imaging, the shutter above the specimen is temporarily closed. In step 2, we adjust the microscope. To avoid further pre-exposure, the illuminating beam is deflected to form a spot of about I pm in diameter, 2 ~m from the crystal. The deflection is adjusted to be parallel to the specimen tilt axis to avoid any focus change. Simultaneously, a 2nd
Model for Bacteriorhodopsin Structure deflection system below the specimen brings the image back to the centre of the viewing screen. Here, alongside the crystal, the defocus is adjusted at a magnification of 150,000 x and a dose rate of 10 e A -2 s -1. This is done by observing the granulation of the carbon support film v/a a television camera and monitor. When the appropriate defocus is found, the magnification is adjusted to 66,000 x where it is known there is no defocus change compared to 150,000 x . The dose rate is then set to 1"6 e A -2 s -t. For this measurement, the final screen is isolated and the beam current is measured by a pico-ampere meter. The loss of electrons, due to secondary emission, is taken into account. The microscope is now ready for recording. In step 3, the image is recorded. The shutter above the specimen is first closed, both deflection systems are switched off, and the film (Agfa 23 D 56) is exposed for 12 s. After the exposure, the defocus is measured directly on the area that has actually been photographed. For this measurement, the beam is increased to 10 e A -2 s -1 and the magnification is set to 150,000x. The film is developed in Kodak D19, full strength for 12 min. For tilting the specimen, specimen holders with fixed tilts are available for different angles. (ii) Spotscan at liquid nitrogen temperature
in Berkeley An efficient spotscan system has been implemented on a J E O L 100B electron microscope equipped with a stable, top-entry stage cooled by liquid nitrogen (Hayward & Glaeser, 1980), a field emission gun electron source, and a computer that controls lens settings and beam position (Downing & Glaeser, 1986; Downing, 1988). Conditions can be set up for searching the specimen, checking or recording an electron diffraction pattern, and focusing and recording the image. Procedures for searching and aligning the specimen are then essentially the same as for the other microscope. The main difference is that, since the J E O L 100B is not equipped with image-deflecting coils below the objective lens, focusing is carried out with the beam deflected on to an auxiliary fluorescent screen, which is mounted in front of the normal viewing screen. Thus, during focusing, the beam is deflected away from the area that is to be recorded on film. Since the deflection is generally not along the tilt axis, the focus is then adjusted by an amount that is predetermined to shift the in-focus line from the front viewing screen to a corner of the photographic film, so that the centre of the film will be at the optimal underfocus. A magnification of about 55,000 × is used both for focusing and recording the image. The high brightness of the field emission gun allows the beam to be focused to a diameter < 1000 A while still retaining the high coherence that is required for good phase contrast imaging. In practice, we usually use a beam diameter of about 1500 A, and step the beam over the specimen in a raster of 15 x 11 points to expose the full photographic film. The exposure time is typically 0"l s/spot, giving a dose of 10 to 15 e A -2 to the specimen. This short exposure time greatly reduces problems of specimen drift, but the long time to record a complete image requires excellent focus stability. In practice, we find that both the high voltage and lens current are sufficiently stable that the focus is essentially constant. (d) Image processing Approximately 20 images were included from each tilt angle range of 0% 20 ° a n d 45 °, with about 10 more at various angles in between (see Fig. 8). All images were
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v
-~'V°IV~-
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Figure 2. Illustration of the contrast variation in an image of a tilted crystal for 1 Fourier component of the structure and how it appears in the Fourier transform of the image. On the left is a function in real space and on the right its Fourier transform. (a) Variation of specimen height across image in a direction perpendicular to the tilt axis. (b) The resulting contrast for 1 Fourier component (i.e. 1 spatial frequency) of the crystal structure. See eqn (3) in Methods, section (d). The Fourier transform shows a corresponding splitting of the diffraction peak. (c) Contrast of the same Fourier component after correction in the computer by multiplication by the estimated tilt transfer function. The Fourier transform now shows the diffraction peak corresponding to the actual structure, with the correct phase. In practice, the correction is applied in the program TTBOX by convolution in Fourier transform space rather than multiplication in real space.
processed using procedures derived from those described (Henderson et al., 1986}. The image data from the untilted and slightly tilted specimens used in the earlier projection analyses (Henderson et al., 1986; Baldwin et al., 1988) are included in the present analysis without further treatment. The new image data from tilted specimens required an extension to the method of image processing as outlined by Henderson et al. (1986) and Henderson & Baldwin (1986). A more detailed explanation of the method is given below. A theoretical discussion of the correction of images for the effects of specimen tilt has also been given by Schiske (1982). (i) Correctionfor tilt For an image of a tilted crystal, the amount of defocus, and therefore the contrast, must vary across the image. Fig. 2 shows a diagram illustrating how the contrast (Fig. 2(b)) for a particular spatial frequency (e.g. one of the Fourier components of the crystal) varies with the height (Fig. 2(a)) of that part of the tilted crystal in the electron microscope. We call the function describing the contrast at a particular distance (p) from the tilt axis and at a single spatial frequency (e) t h e tilt transfer function (TTF (e, p)). This tilt transfer function is an extension of the normal contrast transfer function (CTF), which describes how image contrast varies with spatial frequency in an image from an untilled specimen. The T T F can be derived from the CTF as follows:
CTF(O) = - 2 sinT = --2 sin
AF-~--c,
,
(1)
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where: is the wavelength of electrons (0"037 ,h- at 100 kV), AF is the defocus, normally in the range 0 to 10,000 A, cs is the spherical abberation coefficient (typically 2 ram) and 0 is the diffraction angle of the scattered electron beam in radians (typically 10 -2 at 3 A resolution). I n an image from a tilted crystal, AF varies with position according to the equation: AF - p t a n s + AFo,
(2)
where: AFo
is the defocus at the origin of co-ordinates, taken as the centre of the image, a is the angle of tilt measured from the plane perpendicular to the beam, and p is the perpendicular distance from the tilt axis to the point of interest. p is given by: p -- ~
sin (•-fl),
where: x, y are the co-ordinates of the point relative to the origin, Z is the angle between the line to the point and the x-axis of the digitised image (tan X = y/x), and fl is the angle between the tilt axis and the x-axis. Substitution for AF from eqn (2) in eqn (1) gives:
TTF(O, p) = - 2 sin (cp + yo),
(3)
where c and ?o are constants for given 0, defocus and tilt: C = 2~ (022 tans);
2.[
02
Thus, the image formed by a single Fourier component has a series of equally spaced, sinusoidMly varying bands of contrast parallel to the direction of the tilt axis. Such an image with this varying contrast can be treated so that all areas are restored to having positive contrast. This could be done, in principle, by multiplication of the image, pixel by pixel, by the T T F which, as a function of p, can be calculated for each position x,y on the image. This gives a new image (Fig. 2(e)), which has a contrast of (TTF) 2. The modified image has positive contrast everywhere. Also, parts that had zero contrast in the raw image and therefore contain only noise are multiplied by zero and the noise contribution removed. It can be shown that, for maximum signal to noise ratio, multiplication in the computer by T T F gives precisely the correct weight to the parts of the image with different contrast. The effect of T T F and (TTF) 2 on 1 Fourier component is shown in reciprocal space in Fig. 2. In Fig. 2(b), the actual Fourier component of the structure is split into 2 equal components, separated by a distance proportional to the number of sinusoidal variations in contrast that have occurred across the image for that Fourier component. This is because, in Fourier space, the component is convoluted with the Fourier transform of the T T F given by eqn (3). In Fig. 2(c), the actual peak is restored and 2 wings at half-height are created, separated from the correct position by the same distance as the amount of splitting in the raw image transform. Here, the split peak
has been further convoluted with the Fourier transform of the TTF. The above analysis assumes that the 2 Friedel-related diffracted beams, which combine with the direct beam to form the image, come from the same Fourier component of the structure and are diffracted at equal angles + 0 and - 0 . This assumption is valid in the current analysis. The procedure used would have to be modified if lower voltage electrons were being used or higher resolution being studied, when a pair of spots such as those in Fig. 2(b) would not be at equal distances from the ideal lattice position. An additional problem would arise if the specimen were thicker, when the two diffracted beams would also differ in their amplitude and phase. Although it has not been necessary here, the procedure could be modified to handle both of these eventualities. The convolution theorem of Fourier transform theory offers an efficient procedure for the implementation of this T T F processing. The Fourier transform of the image is convoluted with the Fourier transform of TTF, thus effectively performing the real space multiplication required to go from Fig. 2(b) to Fig. 2(c). Since the T T F is a slowly varying function, it can be described adequately by a smaller matrix (e.g. 150x 150 is adequate for a 6000 A square area of a 60 ° tilted image at 3 A resolution) than that required to sample the image in 1 A steps (this requires 6000 x 6000 pixeis). The convolution is therefore a local operation, using a much smaller part of the image transform than the entire area that would have to be treated if the processing were carried out by multiplication in real space. In earlier work (Henderson & Unwin, 1975), the inefficient real space multiplication was performed directly with an inelegant and less general trick to speed up the computation. The new procedure has been implemented in the form of 3 computer programs, TTBOX, T T R E F I N E and TTMASK, which are described below. TTBOX is the simplest of the 3 new programs. I t corrects the transform with the T T F and determines the amplitude and phase of each spot by examining a box around each lattice point. It performs the convolution correction from an input consisting of the Fourier transform of the image, and a list of values for the parameters involved in the T T F {eqn (3)). The magnification of the image is also needed in order to relate distance in the transform to true spatial frequency. Of these parameters, the spherical aberration (cs) and the wavelength (),), determined by the electron voltage (kV), are fixed and known for the microscope used; the magnification, M, the tilt angle (a) and the azimuth of the tilt axis (fl) are known approximately when the image is taken and can be determined accurately from the foreshortening of the lattice para~meters found in the image transform (Shaw & Hills, 1981); the defocus (AFo), which in general is different for spots in different parts of the transform due to astigmatism, is a function of 3 parameters, AFt, AF2 and ~b, whose values have to be determined during the analysis: AFo = AF 1 cos 2 ( ~ b - ¢ ) + A F 2 sin 2 (~h-¢},
(4)
where AF t and AF2 describe the amount of defocus in the centre of the image in 2 orthogonal directions. ¢ is the angle specifying the direction of AF t relative to the x-axis of the digitized image. Eqn (4) gives the value of AFo that applies to a Fourier component whose direction in the transform is at an angle ~ to the x-axis of the digitized image (Zemlin et al., 1978). The output of TTBOX consists of a list of amplitudes and phases for the Fourier components of the crystal, fully corrected for tilt, defocus and astigmatism, and ready to be merged with data from other images. There
Model for Bacteriorhodopsin Structure are also several useful diagnostic graphical and tabular printouts, which enable the experimenter to judge whether an image is good and whether the processing has been optimized. The amplitude and phase at each lattice point are accompanied by a calculation of the local value of the r.m.s, background in the transform taken from the perimeter of a box surrounding the lattice point, together with an estimate of the resulting error in the phase. We have found it useful to define, for each spot, a parameter indicating spot quality, IQ = 7 (B/A), where A is the background corrected amplitude of the spot and B is the background near the spot. Thus, a spot with IQ = 1 is very good, with signal to noise ratio greater than 7"0, whereas a spot with IQ = 7 is very weak, with a signal to noise ratio of 1"0. We use IQ = 8 to describe a spot whose amplitude is above background but by an amount less than B, and IQ = 9 when the Fourier transform value at a lattice position is less than the r.m.s, background (i.e. A is negative). In the Fourier transform of an image without any specimen present, 65°/o of the data have IQ = 8 or 9 because of the x e -x shape of the distribution of noise (Hayward & Stroud, 1981). (ii) Correction for spatial distortions The above has described how the T T F processing is carried out, but real images also contain spatial distortions. The most important are those arising from stretching and bending of the crystal or the presence of cracks, defects or dislocations of the lattice due to crystalline defects. In the spotsean images, drift over the long period of exposure sometimes produced substantial shifts of the different areas that make up the image. These distortions, as well as those arising from pincushion and spiral distortions of the electron microscope, can be removed by the application of real space-correlation methods (Henderson et al., 1986) based on methods similar to those used previously by others (e.g. Crowther & Sleytr, 1977; Frank et al., 1978; van Heel & Hollenberg, 1980; Crepean & Fram, 1981; Frank, 1982; Saxton & Baumeister, 1982). (iii) Interdependence of defocns and spatial
distortion corrections For the tilted specimens, the problem is that the T T F correction in reciprocal space as well as the unbending correction in real space are both 6ssential and interdependent. After some preliminary experiments, in which the order of the application of the corrections and the amount of iteration was varied, we settled on the scheme shown as a flow diagram in Fig. 3, in which the unbending and the T T F correction were both improved in a cyclical manner. Two further extensions of the TTBOX program were needed in order to achieve this. The functions of these 2 programs, T T R E F I N E and TTMASK, are described below. T T R E F I N E allows the 3 defocus parameters (AFI, AF2 and ~b in eqn (4)) on which the T T F depends to be refined. T T R E F I N E is an iterative least-squares refinement program that maximizes the sum of the TTF-correeted amplitudes of all the spots within a chosen resolution range. The quantity to be maximized is ~'~.A2, where A denotes a TTF-corrected amplitude: A =
.
6A.
opl
apl+
6A . - ap2+
op2
~A
.
- ap3,
op3
(5)
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where Pl, P2 and P3 are the parameters AFI, AF2 and ~b, A 0 is the TTF-corrected amplitude based on current parameters, and 5A/Spl, ~A/Sp2 and 6A/~p3 are the partial derivatives of the TTF-corrected amplitude with respect to the parameters. These are produced by convolution of the image transform with the transform of the partial derivative of the T T F with respect to the parameters. For example, 5A[$pl is calculated using the transform of 5(TTF)/Spl, which is worked out, pixel by pixel, by differentiation of eqn (3). Note that in eqn (3), ?o is a function of AFo, and AFo is specified by the parameters as given in eqn (4). Thus, T T R E F I N E calculates 3 more convolutions in addition to that carried out by TTBOX. The improvement of the defocus parameters is normally continued until the refinement converges. The T T R E F I N E step, used at the end of each pass, can consume a lot of computer time (4 times the number of convolutions and many cycles compared to the single convolution in TTBOX). In the beginning, we tried to estimate the midimage defocus by optical diffraction so that refinement could begin near the correct value. Later, with experience, we simply guessed a reasonable value and tried a series of different starting points, allowing the refinement program to show which defocus values gave the largest number of good spots (those with low IQ) and the highest average amplitude. Typically, the program is able to converge from defocus estimates that are within 1000 A of the correct values. The selection of a starting point further from the correct value than this would cause the refinement to go in the opposite direction and end on a smaller maximum. This is because the T T F is a sinusoidal function and repeats itself exactly at a different height for each spot. 0nly for a single value of mid-image defocus, however, are all spots treated correctly. Care is therefore needed in using T T R E F I N E . Fortunately, in the later steps of merging data from different images, there are many checks that the correct defocus has been determined. T T R E F I N E can also refine the tilt parameters a and fl, but in the analysis reported here these have been left at their initially determined values, which are sufficiently accurate. The program TTMASK, used at the beginning of second and subsequent passes, reads in the Fourier transform of an image and writes out another Fourier transform, corrected for tilt, defocus and astigmatism. I t also carries out masking using circular or square apertures around the position predicted for each spot and leaves the whole area of the TTF-corrected transform inside the mask rather than just the single amplitude and phase at the predicted position, as is done by TTBOX. This allows the data that describe the image distortions to remain in the TTF-corrected transform, whilst removing noise from between the spots. (iv) Flow chart The 3 programs TTBOX, T T R E F I N E and TTMASK are used in the procedure as shown in Fig. 3. The other steps in the scheme use the established set of programs for correcting untilted images for distortions (Hendemon et al., 1986). In the 1st pass through the scheme, the image is treated as if it were untilted and the steps taken are shown on the left-hand side of the diagram. The 1st pass normally resulted in the successful unbending of a strip of the image that extended across the full width in a direction parallel to the tilt axis, but was more limited in the perpendicular direction. For 45 ° tilted images, only about 30% of the area was treated successfully in the 1st pass. The values of the defocus and astigmatism parameters
R. Henderson et al.
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I
Raw dansltometered ........ Image 6000x6000 plxels I
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Fourier transform of raw Image Rrst pass
J
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Fourier transform of current unbent Image
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List of amplitudes and phases i Figure 3. A flow diagram showing the computing steps in the processing of images from tilted specimens. Data sets are enclosed in boxes; programs are in bold type. Light shading behind the program name indicates that the program was developed earlier for the processing of images from untilted specimens; black shading indicates a new program developed for the work with tilted specimens. Broken lines indicate the transfer of refined parameters; full lines indicate data flow. AF1, AF2 and ¢ could then be determined using TTREFINE, and these values used as input to TT~J~SK in the 2nd pass of unbending, as shown on the right-hand side of Fig. 3. The 2nd pass was carried out using the masked TTF-corrected transform of the raw image together with the TTF-correeted Fourier transform of a reference area taken from the unbent image of the 1st pass. By using a tight mask for the Fourier transform of the unbent image,
a very highly averaged reference area is obtained, whilst a generous mask for the Fourier transform of the raw image enables all the distortion information to be included in the analysis. In subsequent passes the procedure cycles as indicated on the right-hand side of the diagram, as the unbending and the accuracy of the defocus parameters improve together. Because the contrast varies so much across the image from a tilted specimen (see Fig. 4(d)), it
Model for Bacteriorhodopsin Structure was not possible to use the weighting procedure described by Baldwin et al. {1988) in the new scheme for tilted specimens, although it would be desirable to do so if it were possible to devise a procedure to pick out which areas of the image were best and therefore should receive the highest weighting.
{e) Parameter determination and data merging The Fourier transform of a 2-dimensional crystal consists of a series of lattice lines continuous in the zdirection. The electron diffraction intensities had been merged previously and intensities fitted to curves along each of these lattice lines using damped sine functions (Baldwin & Henderson, 1984; Ceska & Henderson, 1990). The fitted curves consisted of intensities sampled at intervals of 0-01 A - I along z*. They were converted to amplitudes using the program TRUNCATE (French & Wilson, 1978) from the CCP4t program suite. TRUNCATE converts intensities to amplitudes, taking into account the correct treatment of negative intensities. Phases from the new images were analysed and compared using the program 0RIGTILT. This program enables the phase origin, and/or crystal tilt and/or beam tilt parameters of individual images to be refined as well as data from many images to be merged. Phases from each image were first compared with the phases produced by earlier averaging of 6 A 3-dimensional data from 3 crystal forms at room temperature (Tsygannick & Baldwin, 1987). At 6 A resolution, a knowledge of the beam tilt (Henderson el al., 1986) is not necessary, so this preliminary treatment of the phase data simply involved bringing the data from all the images to the common crystallographic phase origin. Imperfect alignment of the illuminating beam affects the high-resolution components of images (Zemlin et al., 1978). The amount of beam tilt must therefore be determined and its effect corrected for. The determination of beam tilt (2 parameters specifying its amount and direction) for images of untilted and slightly tilted specimens had been done (Henderson et al., 1986; Baldwin et a/., 1988). The determination of beam tilt for the images from tilted specimens required an iterative procedure and was carried out in 2 stages, first for all the 20° tilts, then for the 45° tilts. For the 20° tilts, a merged, list of phases was first produced with the 20° image beam tilts set to zero b u t including the previous correctly treated untilted data. This merged list was then used as the reference for preliminary refinement of beam tilt .by comparing data from each image against data from all the others, excluding itself. Second and subsequent merges, followed by further beam tilt refinement resulted in the iterative convergence of beam tilt and phase origin to, in most cases, unambiguous values with convincing phase residuals, which showed good agreement at resolutions out to 3"5 A (see Table 2). The 45° tilts were then included with preliminary beam tilts derived by comparison with the 20 ° merged data, followed again by iterative refinement. Ten cycles of iterative merging and beam tilt refinement were carried out in this way. The merged list of phases (1.7,922 phase measurements from 72 images including all data with IQ values between 1 and 8) was then combined with the list of amplitudes from electron diffraction and used as input to the program
~f CCP4 is the Collaborative Computational Program in Protein Crystallography.
907
LATLINE (Agard, 1983). LATLINE fits amplitude and phase curves to the non-uniformly sampled data on each lattice line. One of the constraints used in the fitting algorithm is that no electron density should be found outside a specified membrane width profile, set in our case as - 2 1 A to +34 A. This 55 A width is greater than the 45 A minimum stacking distance (Henderson, 1975) seen by X-ray diffraction of dried membranes and was chosen to avoid the possibility of erroneous truncation of density. The profile is centred on a phase origin that is about 6 A from the middle of the membrane because of the asymmetric mass distribution. Each amplitude and phase is accompanied by its own standard error, a, and these were used to calculate l/a 2 weights, which are used by the LATLINE program to calculate the standard errors of the fitted values as well as the best fit. The amplitude data were already adequately sampled (Ceska & Henderson, 1990) at 0"01 A -1. I f new phase data for a lattice line were not measured over any region wider than 0"02 A-1, the fitting of the curve was truncated to the continuous region that was well sampled. The output from LATLINE is then a set of amplitudes and phases that represent the best estimates of the structure factors from all the data. Note that this method of merging makes no use of the image amplitudes, and assumes that the amplitudes and phases are derived from precisely the same structure, even though they are measured by different methods. There is little doubt that these are good assumptions, since the electron diffraction amplitudes are much more accurate than the image amplitudes, and the high correlation coefficients obtained between image and electron diffraction intensities (see Results) shows that, at least for the best images, the structures being observed are the same. Having obtained a complete set of merged amplitude and phase data, 2 final checks were carried out. First, the tilt axes and tilt angles of all the images were refined by minimizing the phase error by comparison with the smooth data from lattice line curves. This was done using 20 to 6 A resolution data only, where the phases are most accurate. Second, the beam tilt on each image was now refined against the merged curves as a final check and to get the most accurate values. These curves were then used to calculate a Fourier map as described below. A 2nd set of curves was fitted with data modified by using individual weights for each spot obtained from calculations of the resolution-dependent fall-off in intensity for 2 directions on each image {Le. parallel and perpendicular to the tilt axis). These individual image weights were used to modify the 1/a2 weights for a 2nd run of LATLINE, and a 2nd set of curves used to calculate a 2nd map. However, the 2 maps are very similar. The weighted map was used in all subsequent work. if) Calculation of map, interpretation and model building Following the calculation of 3-dimensional amplitudes and phases sampled at 0"01 .h.- 1 along the lattice lines, the data were converted into LCF (Labelled Column Format) data files used by the CCP4 crystallographic program suite. All further calculations could then be carried out using standard crystallographic programs. The standard errors on the phases calculated by LATLINE were converted into figures of merit and used as weights in the calculation of the Fourier map. Interpretation of the map and model building into the density were aided by stacks of transparent contoured sections {minimaps) and the graphics program FRODO (Jones, 1978}. Frequent use of the regularization option in FRODO
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R. Henderson et at.
allowed the model to be constrained to a-helices where apropriate. The R-factor between calculated and observed amplitudes, using all electron diffraction amplitude data to 3"5 A resolution and 60° tilts, was 45% for the initial model, which included 10 lipid molecules/asymmetric unit. A final pass of the raw co-ordinates through 50 cycles of Hendrickson-Konnert (Konnert & Hendrickson, 1980) refinement produced a model with good stereochemistry and an improved R-factor (35 % for all data to 3"5 h). 3. R e s u l t s
(a) Electron microscopy and assessment of data Images of tilted specimens, recorded on any of the microscopes used, were examined by optical diffraction. Good images were immediately obvious, showing strong sharp diffraction spots extending to high resolution. By contrast, bad images frequently showed very few diffraction spots. Between these two extremes, there was a great variety of images with all sorts of defects: a thin band of spots due to specimen or image movement during the exposure; very fuzzy spots everywhere, indicating disorder of the specimen during specimen preparation; variability of resolution over different areas of the same micrograph; strong low-resolution spots with nothing visible beyond 10 A. To develop a simple criterion that would allow us to decide which images should be subjected to computer analysis, we endeavoured to estimate the resolution parallel and perpendicular to the tilt axis, and counted the total number of unique spots visible in the optical diffractometer. This latter criterion, the total number of spots visible, turned out to be the most useful, although some images showed such an obvious indication of one good direction and one bad direction, that it was easy to take this into account. However, attempts to make more exact estimates of resolution at the optical diffraction stage turned out to be less valuable. Some of the images that had looked less promising at the optical diffraction step, turned out to contain good highresolution data after computer processing. As a result, we resorted to the procedure of selecting the best images, but also including many that looked less good at the stage of optical diffraction. "This procedure ensured that we did not exclude any good images from the computer analysis, but resulted in the inclusion of some images with very weak highresolution spots. Lattice and image distortions are taken care of by correlation analysis. In Figure 4, for an image of a 44 ° tilted specimen, we show the maps of the correlation peaks obtained during the first pass ((a) and (b)) of analysis and during the last pass ((c) and (d)), following the procedure described in Figure 3 and Methods, section (d). During the first pass, no correction for defocus has been made but, since most of the image is slightly underfocused, an approximate correlation map is still obtained. The bottom left corner of the image shows the lower contrast that is expected for the part of the image that is closer to
focus. In images of more highly tilted specimens such as this, the band of zero contrast, where the specimen height brings a region of the image precisely into focus is often wighin the processed area, together with an area of overfocus on the other side of the band. In Figure 5, we show in resolution zones the average quality of the spots in the Fourier transform of an image after application of the full correction for distortions and defocus. In this case, the image was recorded from a 41 ° tilted specimen using the spotscan procedure. In all zones, and in directions both parallel and perpendicular to the tilt axis, the average diffraction spot is welt above the mean background. Clearly, the lower-resolution data are best and at high resolution, the data parallel to the tilt axis have a higher signal-to-noise ratio than the data perpendicular to the tilt axis. Figure 6 shows, for the same 41 ° tilted specimen as in Figure 5, a representation of all the spots out to 3"5 A and their strengths, indicated by their size and IQ value (1 is best, see Methods, section (d) for definition). Figures 5 and 6 demonstrate the presence of many strong spots in the corrected transform of the unbent image. Further proof that the data obtained from these images arises from the same structure that we see by electron diffraction is shown in Table 1, which shows the correlation coefficient between the intensities (amplitudes squared) of the diffraction spots in the image and the corresponding intensities from the merged electron diffraction patterns (Ceska & Henderson, 1990). The correlation is more than 0"47 at all resolutions and in all directions, except in the intermediate resolution zone in the tilted direction, where the diffraction is always very weak. In some images from 20 ° tilted specimens, the correlation was more than 0"80 in all zones and all directions. However, other images we have included from highly tilted specimens were not as good as this. In the worst cases, correlation coefficients dropped to near zero in the highest resolution zone. In nearly all images, the spots perpendicular to the tilt axis in the higher-resolution zone were the weakest, as would be expected if they were affected most by lack of specimen flatness and beam-induced specimen movement perpendicular to the support film. In all, from about 50 images of tilted specimens that were subjected to computer analysis, about 30% showed peaks greater than 10 in plots similar to those in Figure5 and correlation coefficients Table 1
Correlation coe~cients between image intensities and data from diffraction intensity curves for image 51056 Position relative to tilt axis Resolution (A)
Parallel
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Model for Bacteriorhodopsin Structure fo}
(c)
909 (b)
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Figure 4. The correlation peaks found in a search of the correlation map of image 20945 showing the positions and heights of the correlation peaks at each of the 20,000 unit cells of the crystal present in this image. The image is from a 44° tilted specimen. In the' 1st pass ((a) distortion vectors ( x 10) and (b) peak heights), a large reference area from the centre of the filtered image is used to calculate the correlation map. The reference area can be seen in (b) as the area of highest correlation. In the final pass, ((c) distortion vectors ( x 10) and (d) peak heights), a smaller reference area is used, which is derived from the average of the entire area of the image after corrections for distortions and defocus as defined in the penultimate pass. In this case, maximum correlation peak height is found at the top of the image where the undeffocus is greatest, and almost zero contrast is found at the bottom of the image where the image is almost exactly in focus. Because of the smaller reference area, finer distortions can be seen in (c) than in (a).
(Table 1) greater than 0-2 at the highest resolution, 30~/o showed diffraction peaks detectably above those expected from an image consisting of pure noise, and the remainder, in effect, contributed diffraction information only out to 5"5 A resolution. Phase d a t a from all images were then merged together using the program O R I G T I L T to determine the phase origin and beam tilt parameters. In earlier stages of processing, only the amplitudes of the diffraction spots were used as diagnostics to check t h a t the correct tilt axis and tilt angle were
being used and t h a t the* determination of defocus was optimal: At the stage of running the program O R I G T I L T (Table2), agreement between the phases of each new image and the phases from other images gave a final check t h a t everything had been done correctly during the processing. The phase agreement is shown for the best 45 ° image in Table 2. The fact t h a t all the residuals, even at the highest resolution, are significantly lower t h a n expected for r a n d o m phases (90 ° ) means t h a t . t h e phases from the image itself are good, and t h a t the
R. Henderson et al.
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Figure 5, Areas surrounding the diffraction spots in the Fourier transform of the distortion and defocus corrected image 51056, divided into resolution bands and analysed separately for spots parallel and perpendicular to the tilt axis. The image is from a 41 ° tilted specimen. The tilt axis is inclined at approximately 65 ° to the horizontal X-axis. The plot is centred on the spots and calculated by averaging the diffraction intensity of all 38 spots in each plot at 7 A, all 27 spots at 5-5 A, and all 96 spots in each 3"5 A plot. The peak in the centre of panel 1 (top left) is 2065. Thus, in this image, the average diffraction spot in the highest resolution zone perpendicular to the tilt axis is above background by 1"5 x background. At 7 A, spots are from 150 x to 300 x background. The wings expected from the contrast stripes in the image can be seen best near the edge of the box in the 5-5 A plot for the stronger spots from the direction parallel to the tilt axis. Below 7 A resolution, the wings show up as shoulders to the main peak, and at 3-5 A the wings are off the edge of the plots. combined data from the other images with which they are being compared are good. Thus, Table 2 is a final indication that high-resolution phases in three dimensions have been obtained successfully. Following the merging of phase data into a set of origin-corrected and beam tilt-corrected measure-
ments, s m o o t h curves were fitted along lattice lines to the combined electron diffraction amplitude and image phase data using the program L A T L I N E (Agard, 1983). S o m e typical plots of amplitude and phases after L A T L I N E are s h o w n in Figure 7. Error estimates on the fitted curves are also obtained. At
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Table 2
Phase residual for image 51056 Res rain (A) 100"0 14-0 10"0 8"1 7"O 6"3 5"7 5"3 5"0 4-7 4.4 4-2 4"O 3"9 3"7 3"6 Overall
Res max (A)
Phase residual (degs.)
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Note that this is only one of 72 images, and only spots with amplitude greater than a certain value (given by IQ<~7) are used in the phase comparison. Fig. 8 gives an idea of the total extent of the phase data.
R. Henderson et al.
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this stage, the weight applied to each phase measurement in the input to curve fitting was a function both of the strength of the spot in the image transform (weight oc 1/a 2) and of the quality and strength of the n e a r b y spots from the same image (see Methods, section (e)). This allowed weak spots on good images to contribute more than spots on poorer images t h a t were known from neighbouring spots to have a very low information content. This weighting scheme eliminated subjective judgements of good and bad images and produced a smooth transition both within and between images. Figure 8 summarizes the e x t e n t in three dimensions of the amplitude and phase d a t a used. Because
images were collected mainly near three tilt angles (0 °, 20 ° and 45°), the distribution of experimental phase measurements is not uniform, as shown in Figure 8 where density of shading represents density of sampling by the images used. Nearly all the Fourier components determined by curve fitting up to 20 ° tilt are well measured, whereas those up to 45 ° are noisier with greater phase errors in the fitted curves. I f all phases up to 45 ° were well measured, then 71 °/o of the Fourier components in the threedimensional transform would have been determined. However, some lattice lines did not have phase d a t a t h a t were sufficiently well sampled, because some parts of the three-dimensional
Model for Bacteriorhodopsin Structure 0.3
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The Fourier cut-off resolution was 3"5 A in the horizontal direction but, because of the limited tilt range of the data collection, the highest resolution Fourier term that is possible in the vertical direction corresponds to 5"5 A. A section through the map just below the middle of the membrane, plotted with ten different contour levels, is shown in Figure 9. The entire map in 7 A slices is shown in Figure 10 contoured only at the second level. One of the 7 A slices is shown with the model superimposed in Figure 11. The resolution achieved can be seen by looking at several profiles through different peaks in the map (Fig. 12). The peaks have the expected shape. Figure l2(a) shows a horizontal profile through the largest peak in the map, which is the fl-ionone ring of retinal as explained below, giving a good estimate of horizontal resolution. Figure 12(b) shows a very similar profile through one of the aromatic side-chains (Tyr185). Figure 12(c) shows a vertical profile of the retinal peak, illustrating the lower vertical resolution as well as the presence of almost resolved features about 7 A above and below it. Finally, in Figure 12(d), we show a quite independent way of illustrating the vertical resolution. Purple membrane embedded in phosphotungstate has been studied by Ceska & Henderson (1990), who collected a full set of three-dimensional diffraction data to 3"0 A resolution. A three-dimensional difference map between phosphotungstate-embedded and native purple membrane, using the new phases, shows several large peaks on the two surfaces of the membrane where the tungsten clusters bind. Since the heavy-atoms are small dense features, their profiles can be used to give a direct indication of resolution. The widths of the phosphotungstate profiles confirm the native vertical resolution seen in Figure 12(c). Thus vertically, peaks 7 A apart are just resolved. The full width at half-maximum of each peak is about 2"5 A horizontally and 7 A vertically. These figures imply an effective Fourier resolution of 3"5 A horizontally and I0 A vertically. (c) Interpretation of the three-dimensional map
transform are intrinsically weak, and others were poorly sampled by the random distributions of tilt axes and angles used. Consequently, the curve-fitted data produced 2700 Fourier components with significantly measured phases, representing about half of the data in a full three-dimensional sample: 29 % of reciprocal space representing the 45 ° missing cone is not sampled at all in this set of data. Figure 8 also shows that electron diffraction amplitudes have been measured up to the higher tilt angle of 60 ° (Ceska & Henderson, 1990) representing 87 % of amplitudes. The unphased amplitudes, some of them at exactly the resolution where the 5 A axial diffraction from a-helices is strong, could not be used in the calculation of the map but will be used in the refinement of the model.
The map shows numerous features that are well resolved from the axes of the seven helices. These must be the side-chains of bulky aromatic amino acids such as Phe, Tyr and Trp as well as the retinal. In particular, the densest peak in the map (Figs 9, 12(a) and (c), and 13(a)) is in exactly the position expected for the p-ionone ring of the retinal, based on the position of the ring label peak in the neutron diffraction difference map (Heyn et al., 1988). Vertically, the peak is about 6 A below the centre of the membrane judged from the positions where density begins on the top and bottom sections of the map. There is therefore no doubt that this peak is the /Y-ionone ring. Above and below the fl-ionone peak (height 10 contours) are almostresolved peaks of half the height (5 contours). Most
R. HendersoT~ et al.
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Figure 9. A section through the map parallel to the membrane plane at the level of the fl-ionone ring of the retinal, just below the centre of the map. There are 10 equally spaced contours with the maximum density in the ring of the retinal.
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Figure 12. Profiles through several identified features to estimate horizontal and vertical resolution. (a) The retinal ring in the horizontal direction. (b) Tyr185 in the horizontal direction. (c) The retinal ring in the vertical direction. (d) Phosphotungstic acid embedded-glucose embedded difference map peak in the vertical direction.
of the other well-resolved peaks are also of height about five contours, whereas the density at the centre of the helices on average rises to eight or nine contours. Noise was one to two contour levels as judged by looking at the map in regions above the ends of the helices but within the membrane profile. We looked first for density connecting the helices at the top and bottom of the map. Regarding the connectivity at the top (Fig. 10), we find that the density can b e followed upwards from each helix until the density from an adjacent helix either merges with it, or the densities from the two helices get so close together and at the same time so far from other helices that the connectivity of the~polypeptide in each region is in little doubt. Similarly, for the bottom sections of the map, where much larger and denser features are present that are quite clearly not part of the seven vertical helices, the density seems to connect pairs different" from those joined at the top. While no connection in the map is unequivocal/the fact that the six indicated connections are self-consistent confirms that the seven helices have the connectivity shown in Figure 14. The helices without a connection, one each at the top and bottom of the map must then be the amino and carb0xyl termini. It is known that the amino terminus is at the bottom of the map, and corresponds to the extracellular surface from biochemical sidedness experiments (Gerber et al., 1977) and electron diffraction of membranes with known orien-
tation (Henderson et al., 1978; Hayward et al., 1978). Hence, the helices in Figure 14 can also be labelled A to G according to their position in the sequence as shown. The connectivity and helix assignments are the same as those suggested previously (Engelman et al., 1980). We can then look to see whether the positions of the bulky aromatic side-chains in the amino acid sequence correspond with the positions of the features that are well resolved fromthe main density of the helices. We looked first gtj helix F where the residues Trp182, Tyr185 and Trp189 are the only aromatic residues in the bottom half of the helix near the retinal ring. The two tryptophan residues are seven residues apart and therefore must be almost vertically above one another separated by 10'5A; Tyr185 must be about 60 ° anticlockwise from the tryptophan residues and halfway between them vertically. If we orient the helix so that the nearly unresolved features above and below the retinal ring represent the indole moieties of the tryptophan residues, there is a density in precisely the right place for Tyr185, which would place the tyrosine side-chain more or less parallel to the aliphatic part of the retinal. Figure 13 shows stereo views of our interpretation of these two features in the map. In Figure 13(a), the fl-ionone ring of the retinal is barely resolved from the side-chain of Trp189 below it. The side-chain of the residue above the ring shows up as a shoulder in the density. Figure 13(b) also shows that the density in helix F that we assign to Tyr185 extends downwards and becomes closer to the adjacent helix G, suggesting the presence of another side-chain from helix G at this lower level. From the sequence, Phe208 is the only aromatic residue in the lower half of helix G. Our interpretation of Figure 13(b) is therefore that the side-chains of Tyrl85 and Phe208 appear as a single elongated peak joining the two adjacent helices together. One consequence of the assignment of the side-chain of Phe208 to the lower half of the density shown in Figure 13(b) is to enable Lys216, connected to the retinal, to be positioned 12A higher than Phe208 and about 80 ° anticlockwise from it. This puts Lys216 in the correct position to form a Schiff base with the aldehyde of the retinal, assuming the retinal to be tilted at about 20 ° to the plane of the membrane, as shown by linear diehroism measurements (Heyn et al., 1977). Similar, clear-cut interpretations of the densities in the lower halves of helices C and E can be made. In each case, there are aromatic side-chains in the sequence and corresponding densities in the map; Trp137 and Trp138 for helix E, and Tyr83 and T+p86 for helix C, as well as a few others. 'Helix B contains rather a lot of bulky residues and the map contains a number of peaks. Consequently, there were two possible orientations that fitted the density, with either Met56 or Tyr57 directed towards the environment of the Schiff base. Since Tyr57 is conserved in all the members of the bR family (see Fig. 19) and is known from mutagenesis to affect the rate of reconstitution of
Model for Bacteriorhodopsin Structure
917
(
/ \ /
I
/ b
%%.I. Y ~. ~)llj
. . . .
,,..
!
,..,
~
fi
I
',
i ~
.
/"4
.\ '
!
'1
/
.I
; I
i
'
/
i
r
.
.L
A
\ a,
i¸
\ ;", ( l
'1
!
• \.
\.
.
,y
f,
J
Figure 13. Stereo view of 2 parts of the map to illustrate the poorer vertical resolution and the results of our efforts to interpret the map. (a) The retinal ring viewed parallel to the membrane showing Trp189 and Trp86. (b) The unresolved density for Tyr185 from helix F and Phe208 from helix G.
apo-membrane with retinal (Mogi et al., 1987), we decided that the second orientation, with Tyr57 pointing inwards, must be correct. Helix A, which is knowr~ to be less important in the functioning of bR (Khorana, 1988), contains two tryptophan residues at positions l0 and 12. We fitted these, as well as Met20, Tyr26 and Phe27, into features that seem unique and that result in an orientation in close agreement with that determined by Popot et al. (1989) using neutron diffraction of
membranes containing leucine-labelled helices A and B. The remaining helix D contains no bulky aromatic side-chains and the map showed no clearly resolved densities. As a result, the orientation of this helix is the least certain part of our interpretation. We simply placed the helix in the density to maximize the correlation of the positions of the five glycine residues with the apparent absence of bulges along the main helix density, and with. A s p l l 5
918
R. Henderson et al.
(b) Figure 14. Overall chain trace. (a) A diagram showing helices as solid rods; (b) ribbon diagram following the backbone of the polypeptide. Helix A, on the left, is connected to helix B at the top; helix B is connected to helix C at the bottom; helix C to helix D at the top; helix D to helix E at the bottom; helix E to helix F at the top; helix F to helix G at the bottom. The N terminus (nt)is at the bottom at the extracellular surface; the C terminus (ct) is at the top at the cytoplasmic surface. Both diagrams show the retinal and its connection to Lys216. oriented inwards, towards and above the retinal ring. This orientation also brings the other two conserved residues in helix D (Fig. 19), Metll8 and Gly122, into the environment of the retinal ring. Thus, the positions of the residues of helix D, while plausible, are not determined with certainty. Fortunately, the helices forming most of the environment of the retinal, B, C, E, F and G, are the most unambiguous. Figure 11 shows the superposition of the atoms on the density in the 7 A slice of the map at the level of the retinal, according to the interpretation given above, after model building and refinement. The final part of the interpretation that gives us confidence comes from the consequence of the identification of well-resolved features in the map with aromatic side-chains in the sequence. We can build each helix upwards and downwards from the assigned aromatic residues and ask whether the remaining lengths of polypeptide chain are consistent with the observed hydrophilic inter-helix links, some of which are small and tight (e.g. CD)
and others of which are much longer (e.g. BC). Considering that the vertical resolution is poor and therefore that the exact conformations of the links are harder to see, there is an excellent correspondence within one or two residues between the observed density in each link and the length of sequence expected from side-chain assignments. In particular, the weaker densities for the three links at the top correspond to extended polypeptide chain, whereas the much denser features at the bottom of the map have bigger stretches of polypeptide to fit into them. Indeed, all three Iinks at the bottom have a density and length that suggest that short stretches of horizontal ~-helix are required to fit the required number of amino acid residues into the density that is present. The remainder of the map was interpreted by building up and down from identified regions using the bulges t h a t protrude from the helices to position the side-chains from smaller amino acids. This served to keep the helices in register, and should result in a correct interpretation, except where there
Model for Bacteriorhod~sin Structure
919 CYTOPLASM
A
B
C
D
E
F
EXTRACELLULAR G
Figure 15. A view of each helical section of the main-chain backbone. The cytoplasmic side is at the top. Clear kinks in helices B, C and F are seen at the positions of Pro50, Pro91 and Pro186. Helix G also seems to be bent near Lye216, whereas the other helices A, D and E are relatively straight. The viewpoint for each helix has been selected to show best the deviations from straightness. Helices B, C and D are viewed parallel to Y. Helices A, E; F and G are viewed parallel to X*. Fig. 9 shows these directions with respect to the 7-helix plan view.
is a possibility of a kink such as might exist at the proline residues in helices B, C and F. Fortunately, this does not seem to be a problem, and the bulk of the structure is unambiguous, including most of the retinal binding site and the residues that line the proton channel near Asp85 and Asp96. No density is seen in the map after residue 225 at the C terminus, nor before residue 8 at the N terminus. (d) A model of the structure of bR The first problem we had in building the detailed model into the density was the conformation of the retinal. The map shows the position of the Schiff base approximately in the centre of the molecule and the ring about 5 A lower towards the extracellular surface, but the resolution cannot show the methyl groups. This gave us the freedom to choose the all-trans, 6-s-trans conformation for retinal
(Harbison et al., 1985; van der Steen et al., 1986) with the plane of the chromophom nearly perpendicular to the membrane plane (Earnest et al:, 1986) and the methyl groups upwards (Lin & Mathies, 1989). The data presented by Lin & Mathies (1989) on the linear diehroism with vitamin A2, which has an extra double bond in the ionone ring, as chromophore gives an unambiguous indication of the methyl group direction when coupled with the definite determination of the vertical position of the retinal obtained from our density map. We themfore built the retinal as shown in Figures 14 and 16. The amino acid side-chains were built into the density as well as possible considering the relatively poor vertical resolution. Most of the Phe, Tyr and Trp side-chains in the bottom half of the helices are well-determined in position but not necessarily in orientation. The vertical resolution does not allow the orientation of side-chains to be determined,
V| 17 S6
')uI
Figurv 16. Stereo view of the retinal environment viewed from the cytoplasmic surface. The positions of residues Trp182 and Asp115 are slightly ambiguous. Nevertheless, both are very close to the ~-ionone ring.
R. Henderson et al.
920 Table
3
Helix parameters Helix A
B
Residues 10-32 38-62
C
80-101
D E F G
108-127 136-157 167-193 203-227
Number
Residue at same level as retinal ring
23 25 22 20 22 27 25
16 56 85 117 139 185 210
The residue number at which each helix terminates is uncertain at the top and bottom of each helix by at least 1 residue.
though sometimes the stereochemistry dictated the orientation. For example, the position of the sidechain of Tyr185 suggests that its ring plane must be parallel to the plane of the extended polyene of the retinal and therefore roughly vertical. Similarly, the rings of Trpl37 and Trp138 are likely to be vertical in orientation. The top parts of the density of most of the helices are weaker and suggest that the structure is intrinsically less well ordered so that positions of side-chains in the top one-third of the helices are less certain. Similarly, although the linking regions have reasonable density and a plausible model can be built, we cannot be so sure that interpretation of the links is not out by one residue in either direction. As mentioned above, the interpretations of the top half of helix F and all of helix D are also less certain. Nevertheless, we are confident of the interpretations shown in Figures 14, 15 and 16. Figure 14 shows two representations of the overall chain tracing. Figure 15 shows different views of the seven helices selected to show up the slight kinking at the three proline residues that are approximately in the centre of helices B, C and F. Helix G seems also to be slightly bent near Lys216. The other three helices are generally straighter. Figure 16 shows a stereo view of the retinal and its environment together with those side-chains that we are most certain about.
Retinal makes contact with Trp86, Trp138, Trpl82 and Trp189. Trpl37 is not in contact with the retinal. T y r l 8 5 and probably Tyr83 are in contact with the retinal, as are Thr89 and Thr90. Altogether, with a slight uncertainty because of the resolution and possible ambiguities, 21 amino acid residue~ in all (Table 4) form the retinal binding pocket. Having now described the retinal binding site, we turn to the residues that may be involved in forming the lower part of the proton channel. The position of Asp212 is such that Tyr57, Tyr185 and Trp86 are its closest neighbours, suggesting that the side-chain is stabilized by hydrogen bonds to the two tyrosine hydroxyl groups and the indolyl NH of Trp86. No density is resolved for Asp85, but the interpretation of the whole helix places Asp85 at a similar distance from the Schiff base as Asp212. Arg82 is below Asp85 and its side-chain could be placed either upwards near Asp85 or downwards away from Asp85, or could move between the two positions. Asp212 is stabilized by more interactions than Asp85. Overall, the environment below the Schiff base, seen in Figure 16, shows a fairly open structure with sufficient room for a reasonable number of water molecules and a constellation of very hydrophilic residues that would be accurately described as forming a hydrophilic and charge-rich channel between the Schiff base and the extracellular surface. In contrast, the region above the Schiff base looks like a narrow, close-packed and hydrophobic region with the single exception of the side-chain of Asp96, which is positioned l0 to 12 A above the Schiff base and 6 to 8 A from the end of helices G and C. Since it is known that Asp96 is the proton donor in the second half of the photochemical cycle in which the Schiff base is reprotonated, the region between Asp96 and the Schiff base must be the upper half of the proton pathway or channel. Thus the cytoplasmic half of the channel is narrow and hydrophobic, whereas the extracellular half of the channel is wider and hydrophilic. Residues lining the proton channel are shown in Figure 17, again with slight reservations because of the poor resolution. More
Table 4
Rexidues that line the retinal pocket Helix
A
Above
B
D
E
F
G
Val49 I~u93 Thrg0
retinal
Level with retinal
C
Met20
Below retinal
AlaS3
Trp182 Met145 Asp115
Thr89 Tyr57
Trp86 Asp85
Serl41 Trpl38
Tyrl85 Pro186
Lys216
Met118 Gly122
Trp189
Asp212
Note: 10 of these residuesat the Schiffbase end are in commonwith Figure 17 becausethey also line
the proton channel.
Model for Bacteriorhodopsin Structure
Cytoplasmic surface
Upper channel
Immediate retinal environment
Lower channel Extrocellular surface
Q
= Retinal binding pocket
Figure 17. Residues lining the proton channel.
precise information on the channel sizes and space available for water molecules should await refinement. Of 21 residues, listed in Table 4, close enough to the retinal to be in probable van der Waals' contact, 13 are identical in hR and 17 in sR. Overall, in the retinal binding site 62 ~/o of the residues are identical in all three proteins, whereas in the rest of the molecule the conservation over all three is only I0~/o . Again this gives us confidence that the interpretation is correct. In the proton channel (Figs 17 and 19), similar comparisons between bR, hR and sR are interesting. In the narrow upper channel, changes in hR make the size of the protein side-chains smaller by the equivalent of five methylene groups, whereas in sR the protein side-chains are larger by eight methylene groups. This suggests that the hR channel, for chloride ions, is larger, whereas in sR the channel must be narrower, and may be entirely blocked. 4. Discussion (a) High-resolution electron microscopy We have demonstrated that it is possible to record and analyse high-resolution images of highly tilted specimens of two-dimensional crystals. These images can then be used to determine the structure of, in this case, a membrane protein in atomic detail. The map we have obtained approaches a resolution typically obtained in X-ray diffraction analyses of protein crystals. More data are being
921
collected. This will give a map improved both in resolution, particularly in the vertical direction, and in noise level. The reason we are able to interpret the present map with some confidence is because the large amount of a-helical secondary structure makes interpretation easier, and because the large body of available structure-function correlations from mutagenesis confirms that the identifications of the features are all reasonable. Without these two valuable supports, we would need to continue to process more images before being able to interpret the density confidently. Although the best images we have obtained contain excellent contrast at all resolutions and in all directions, they are still far from attaining the best that is, in principle, possible. The best image of native purple membrane so far obtained is an image of an untilted specimen taken at liquid helium temperature in Berlin (Baldwin et al., 1988). I t shows an average peak-to-background ratio of diffracted intensity in the 3"5 to 5"5 A resolution range of 22 and significant power beyond that out to 2.8 A. As shown by Henderson & Glaeser (1985), even this is less than a good image should have with a perfect microscope and specimen. In the present work, using tilted specimens, the best peak-to-background ratio in the 3-5 to 5"5 A resolution range is 10 near the tilt axis, 6 perpendicular to the tilt axis for 20° tilted specimens and 2"5 perpendicular to the tilt axis in a 41 ° tilted specimen (see Fig. 5). The best image of a tilted specimen was taken at Berkeley using the spotscan procedure with highly coherent illumination (i.e. field emission gun). The remaining tasks that need to be tackled to make the use of image phasing a robust and reliable method in electron crystallography include a better understanding of specimen preparation; the behaviour of carbon support films is very variable. We need to understand why some specimens lose their crystallinity on some support films, and why some two-dimensional crystals can make flatter and smoother specimens than others. There is also the need to overcome the problem of specimen or image movement or blurring which, especially in tilted specimens, causes a great loss of high-resolution contrast. This is due either to physical movement of the specimen as it is damaged by the electron radiation or to build-up of charge during the exposure to electrons. In tilted specimens, the movement or blurring is often worse in the direction perpendicular to the carbon film, making good images of tilted specimens much more difficult to obtain. Clearly, spotscan (Henderson & Glaeser, 1985; Downing & Glaeser, 1986; Bullough & Henderson, 1987; Downing, 1988; Zemlin, 1989) provides a substantial improvement but at least one more technical advance would help. In any ease, it is clear that specimen cooling, preferably to liquid helium temperature, and the use of an illuminating beam that covers only a small area of the specimen at any instant (spotscan) both produce valuable improvements in image quality. For images of tilted specimens, in addition, the coherence associated with the
922
R. Henderson et al.
electron beam from a field emission gun produces an envelope function that extends to high resolution even for regions of the image that are highly defocused. We conclude that no currently available microscope is perfect. The time is therefore ripe for a fresh effort to develop a microscope with all the features we now know are essential for this type of work. These are a stage temperature preferably near that of liquid helium, good positional stability, very high vacuum to avoid contamination of ice on the specimen, computer control of all microscope functions to allow spotscan imaging, a field emission gun for superb coherence, and ease of use in the normal operator environment provided by one of the major electron microscope manufacturers. A higher voltage than that presently available, say 200 or 400 kV would also be an advantage. This would allow the investigation of the remaining specimen problems that at the moment are difficult to untangle from the other problems that sometimes occur with the temperamental prototype cold stages and elderly microscopes that we have been using. Once there is a reliable and easy to use microscope, and the remaining problems of specimen movement and image blurring are overcome, it should be possible to record images regularly that are better than any yet obtained. This would then turn the technique we have been using into a routine and quick method, able to be used on many more difficult specimens, eventually including non-crystalline molecular assemblies. The future of high-resolution molecular structure determination by electron microscopy will then be even brighter than it is already. (b) Structure of bR: correlation with other work The vertical position of the retinal with the Schiff base in the centre of the protein and the ring about 5 A lower and nearer the extracellular surface gives a tilt angle of roughly 20 ° ( _ 10°) out of the membrane plane, in agreement with most other measurements, which range from 15° to 25 ° (Heyn et al., 1977; Earnest et al., 1986; Lin & Mathies, 1989). Previous attempts to determine the distance of the retinal ring from either membrane surface ~ have concluded that it is indeed nearer the extracellular surface by chemical cross-linking (Huang et al., 1982), by a second harmonic interference technique (Huang & Lewis, 1989), and by energy transfer (Leder, R. O., Helgerson, S. L. & Thomas, D. D., unpublished results). Otomo et al. (1988) have suggested that the ring is nearer the cytoplasmic surface, but Lin & Mathies (1989) and Leder et al. (unpublished results) explain the results presented by Otomo et al. (1988) as being due to higher energy transfer resulting from specific adsorption of their probes by the negatively charged cytoplasmic surface, thus resolving their disagreement. Huang et al. (1982) found that their ring-labelled retinal reacted with residues Ser193 and Glu194, whereas we find that the ring is some l0 A above Trp189~ level with
Pro186. However, if the retinal is allowed to swing downwards, pivoting about the Schlff base, then it can reach residues 193 and 194, which are ideally placed to react with a bulky ring-labelled retinal that is perhaps too big to fit into its normal binding site. Linear dichroism measurements before or after reaction to determfine the angle of the azidophenylretinal could test this explanation. Our position for the retinal ring, distant from residues 193 and 194 also explains why replacement of Gtu194 by Gin has no effect on the bR spectrum (Hackett et al., 1987). The neutron diffraction results of Hauss et al. (1990), which also indicate a non-central position for the retinal ring without specifying which side, are also in agreement. Lin & Mathies (1989) present the only measurement that determines in which direction the methyl groups point. We have assumed this is correct in building our model, since the resolution of our density map is too low to see the methyl groups directly. The position of the retinal viewed in a direction perpendicular to the membrane plane is in excellent agreement with recent neutron diffraction studies (Heyn et al., 1988). The polypeptide chain trace agrees with that previously deduced as being the most probable by Engelman et al. (1980), and subsequently used in nearly all efforts to present coherent accounts of a variety of data (Khorana, 1988; Oesterhelt & Tittor, 1989; Popot et al., 1989; Lin & Mathies, 1989). Of the arguments used by Engelman et al. (1980), the one that argued that the structure should contain the shortest links able to connect the ends of the low-resolution helix density can now be seen to be the most powerful, since the linking regions that contain longer stretches of polypeptide are now found to be physically compact, possibly forming short stretches of horizontal ~-helix. Similarly, the lower density found in the positions occupied by helices A and D was helpful. However, the suggestion that there would be a salt-bridge between Lys172 and Aspll5, and another between Arg175 and Asp96 is now seen to be wrong. The orientation of helix F does not allow it. With Tyr185 and Pro186 directed towards the centre, the side-chains of Lys172 and Arg175 are on the upper surface of the tilted helix, directed outwards from the centre of the molecule and to the right in Figure 14. Although they are not really high enough on helix F to be fully exposed to solvent, we presume they are able to reach the polar lipid headgroup region. This position for Arg175 seems consistent with the observation that the mutant R175Q has a tenfold slower folding rate (Stern & Khorana, 1989) and that Arg175 may have a role in correctly orienting helix F during folding. Neither the lipids nor the side-chains in this topmost part of the structure are sufficiently ordered to see them. The C terminus from residue 225 onwards is disordered, as shown previously (Wallace & Henderson, 1982). Agard & Stroud (1982) claimed to be able to reveal the link regions using a density modification procedure on much lower resolution data from Henderson & Unwin {1975). Some of the extra
Model for Bacteriorhodopsin Structure features that they found occur in positions where we now find density but others do not. For example, the link between the tops of helices C and D, which shows up as the clearest link in our map, was not observed, whereas the E F link, which was the only density at the top of their modified map, is in fact absent. Previous indications of link regions in the experimental maps (Leifer & Henderson, 1983; Tsygannik & Baldwin, 1987) are now confirmed at higher resolution in the same places as seen before. In their vertical positions across the membrane, the beginnings and ends of the helices (Table 3 and Fig. 19) are not substantially different from many previously proposed arrangements (e.g. Ovchinnikov et al., 1979), though perhaps helix D is physically positioned rather lower than in most proposals and helix F considerably earlier in the sequence and lower physically than in some (e.g. Engelman et aL, 1982; Dunn et aI., 1987). Nearly all the information from chemical labelling studies or from proteolytic enzyme and antibody accessibility studies, summarized by Ovchinnikov et al. (1985), is in good agreement. Within the plane of the membrane, neutron diffraction studies of specifically deuterated labels have shown the position and orientation of helices A and B (Popot et al., 1989), electron diffraction suggested a C terminus position (Wallace & Henderson, 1982), and neutron diffraction of deuterated, phenyl-isothiocyanate-labelled membranes suggested a position for Lys41 (Seiff et aI., 1986). Only the last of these studies is irreconcilable with the present model. Even an extended sidechain with a bulky phenyl group label cannot reach from the present position for Lys41 in the AB link to the peak in the difference map of the phenylisothiocyanate-labelled membranes. The retinal environment includes four tryptophan residues as found in energy transfer experiments (Polland et al., 1986). Three of these, Trp182, Trp189 and Trp86, were identified by Mogi et al., (1989b) on the basis of results from site-directed mutagenesis. Two detailed models for the environment of the retinal have been proposed, based on these and other results (Rothschild et al., 1989a; Lin & Mathies, 1989). Both propose that Trp182 and Trp189 sandwich the central polyene of the retinal, as also suggested by Oesterhelt & Tittor (1989). In addition, Rothschild et al., (19895) include Trp86 as part of the proposed retinal pocket. The position that we find for Trp182 is indeed above the centre of the retinal, whereas Trp189 is below the fi-ionone ring. The sandwich is formed by Trp182 above and both Trp86 and Trp189 below the retinal. The positions that we find for the residues of helix F are certain, since they are determined by the position of the retinal ring, the position and tilt of helix F and the interpretation of the resolved densities for the bulky aromatic side-chains. In the model proposed by Rothschild et al. (1989a) and Braiman et al. (1988a,b) based on vibrational spectroscopy of sitedirected mutants, Tyr185 is placed close enough to the Schiff base to interact directly and they propose that the tyrosine hydroxyl is deprotonated and
923
forms a counterion at the beginning of the photocycle. In our structure, the tyrosine hydroxyl group is 6 A from the Schiff base, so this is not possible without considerable and unlikely helix movements during the photocyele. Instead, Tyr185 seems to form a hydrogen bond to the negatively charged Asp212 side-chain, producing an indirect interaction with the Schiff base. The fiat ring of Pro186 is in contact with the fl-ionone ring of the retinal, and could explain the observed effects of replacement of Pro186 by giycine, alanine, valine and leucine residues (Mogi et al~, 1989a). ~I~he arrangement proposed by Lin & Mathies (1989) also comes close to our findings, although the rotation of their helix F by 90 ° removes the close contact between Tyr185 and the extended polyene of the retinal that we find. In summary, the previously proposed models have turned out to be remarkably good, especially where they have been guided most closely by data. (c) Proton pumping In Figure 18, we show a diagram indicating the locations of Asp96, Asp85, Asp212 and Arg82, which line the proton channel, and their relation to the retinal ancl Lys216. These residues are the most important in the light-driven proton pumping. Tyr57 and Tyr185 are also close to the active site and both seem to make hydrogen bonds with Asp212. However, Mogi et al. (1987) have shown that the mutants Y57F and Y185F are still substantially active as proton pumps, whereas mutations to Asp85 or Asp96 or, to a lesser extent, Asp212, inhibit proton pumping (Mogi et al., 1988). Stern & Khorana (1989) have further shown that Arg82 affects the pK of one or both of the aspartic acid residues but, apart from this general effect, does not affect proton pumping. A minimal model for proton pumping would therefore involve the three aspartic acid residues and the Schiff base. The role of Asp96 has been established recently as that of the proton donor in the latter part of the photochemical cycle when the Schiff base, deprotonated at M412 is reprotonated to form N56o (Butt et al., 1989; Gerwert et al., .1989; Tittor et al., 1989; Holz et al., 1989; Otto et al., 1989). Its position 10 to 12 A above the Schiff base and the fact that it is protonated prior to M (Braiman et al., 1988a; Gerwert et al., 1989) is entirely consistent with this function. We assume that there must be at least one or two structured water molecules in the otherwise very hydrophobic channel above the Schiff base. The side-chains of Asp85 and Asp212 are approximately equidistant from and 4 A below the Schiff base and, since they are both unprotonated in bRs7 o, could be considered equally good Candidates for being counterions to the Schiff base in bRsTo (Braiman et al., 1988a; Gerwert et a/., 1989). However, on release of the Schiff base proton to form M412, we presume that only one of the two carboxyl groups acts as a proton acceptor. On the basis of the more hydrophilic surroundings of Asp212, we think that Asp85 is the more likely group to a c t as the
924
R. Henderson et al.
Figure 18. Artistic impression showing the relationship between the key residues Asp85, Asp96, Asp212, Lys216 and Arg82, and the retinal binding site, the proton channel and overall molecular boundary for the ground state of bR (bR57o). The cytoplasm is at the top of the diagram.
proton acceptor, consistent with the results of Mogi et al. (1988) that the mutation of Asp85 to asparagine has a much larger effect on proton pumping than the same change to Asp212. The role of Asp85 as proton acceptor in the transfer from the Schiff base to the extracellular surface is supported by the interpretation of Fourier transform infra-red measurements, which suggest that Asp85 is unprotonated in bR, K and L and protonates in M (Braiman et al., 1988a). It is supported by the fact that the D85E mutation affects the rate of formation of M (Butt et al., 1989) but retains some pumping activity, whereas D85N is completely inactive. Finally, Asp212 is unchanged whilst Asp85 is changed to Thr in hR, which pumps chloride instead of H + andhas no deprotonated M intermediat6: We therefore favour Asp85 as the site to which the proton is transferred from the Schiff base in spite of the fact that Braiman et al. (1988a) and Gerwert et al. (1989) interpret their Fourier transform infra-red measurements to indicate that both Asp85 and Asp212 become protonated in M. It should also be noted that this open arrangement on the extraceltular side of the Schiff base is consistent with the rapid proton exchange at the Schiff base observed by nuclear magnetic resonance spectroscopy and other methods (Harbison et al., 1988~. Similarly, the collection of charged residues in this region is consistent with the idea of a complex counterion for the protonated Schiff base that has.been inferred from nuclear magnetic resonance d a t a (De Groot et al., 1989).
In a minimal mechanism of proton pumping, shown schematically in Figure 20, we assume that the isomeric state of the retinal as derived from interpretations of resonance Raman data is correct (Smith et al., 1983, 1984, 1986; Fodor et al., 1988). It is necessary to propose transient protonation changes only to Asp96 and Asp85, leaving Asp212 permanently deprotonated and Aspll5, situated near the fl-ionone ring, permanently protonated. On isomerization of the retinal, the Schiff base pK becomes lower when it is connected via Asp85 to the extracellular surface, and subsequently returns to a high p K when it is connected v/a Asp96 to the cytoplasmic surface. The corresponding proton movements result in pumping. Clearly, Asp212 and A s p l l 5 would still be necessary in providing the correct charge environment at both the Schiff base end and the fl-ionone ring end of the chromophore. Structural changes in the different intermediates, which probably involve changes in Asp212 and Aspll5, must affect both the absorption spectrum of the retinal and the effective pK of the Schiff base. The effective p K values of Asp85 and Asp96 may also change but this is not essential to the discussion. In the minimal mechanism, the all-trans to 13-cis isomerization at the K intermediate first moves the protonated nitrogen of the Schiff base to a new environment more distant from the side-chain of either Asp85 or Asp212 or both. A conformational change in the protein, which could be quite small (e.g. it might include rotation of the carboxyl group
Model for Bacteriorhodopsin Structure A
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Figure 19. Our alignment of sR, bR and hR sequences with the helical segments outlined by a box and the correct relative heights indicated. The height of the retinal ring is at the level of Tyr185 and the cytoplasm is at the top. It can be seen that a very large proportion of the conserved residues line the retinal binding pocket (diamonds) but there is less conservation of residues in the proton channel (circles).
of Asp85 about its =-fl bond), to form L brings the Schiff base closer to Asp85 and into an environment to favour proton transfer in the formation of M. In the transition from L to M, a proton is released at the extracellular surface (Lozier et al., 1976), so the effective pK of both the Schiff base and Asp85 must be low. In order that the protonated Asp96, which has a high pK, can then transfer a proton to the Schiff base to form N, a second structural change in the protein must follow before the transition from late M to N so that the p K values and accessibility between Asp96 and the unprotonated Schiff base create conditions that favour the second proton transfer. Here, we call early M the structure formed by proton transfer from the Schiff base in L. This discriminates between the low p K state after proton transfer and a later high pK M state, which is required before reprotonation can occur. This late M intermediate is the form that would be observed kinetically, whereas early M is likely to be a shortlived, kinetically non-observable and therefore hypothetical intermediate. Finally, Asp96 must be reprotonated and the retinal must re-isomerize to form O~40 and convert back to hR. At least three
distinct protein structures are involved, best characterized by the K to L transition, the early M to late M transition and the 0 to b R return to ground state though, in practice, it might be more complex. For example, there is beginning to be evidence (Otto et al., 1989) that the reprotonation of Asp96 is necessary before the re-isomerization of the retinal to form 0. This would imply a further change of structure between N and O. At any of these stages, but it would be expected particularly in L and early M, the retinal may be in a strained out-of-planar conformation, which would certainly lower the Schiff base p K (Schulten & Tavan, 1978; Fahmy et al., 1989). Apart from distortions to make the retinal non-planar, two other effects seem likely to play a role in lowering the p K of the Schiff base in L and early M. The environment of the Schiff base may be more hydrophobic than it is in bR, N and O, and the side-chain of A s p l l 5 may be placed further from the ring of the retinal. ~ this discussion, protein structure changes play an important role in the mechanism, as they do in the C-T switch proposal of Fodor et aL (1988}, though the points at which changes might occur are
926
R. Henderson et al.
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(b)
Figure 20. A diagram to describe the 4 proton movements in the photocycle of bR. The only observed structure is bR. The others with the 13-cis isomerization of the retinal are speculations in which the isomeric state of the retinal is taken directly from interpretations of resonance Raman data (Smith et al., 1983, 1984, 1986; Fodor et al., 1988). It is proposed that the protein takes up at least 3 distinct conformations; (1) as in bR and K; (2) as in L and early M to facilitate the proton transfer to Asp85 to form M; (3) as in late M~and N to facilitate the proton transfer from Asp96 to form N. The Schiff base pK must be low in L and early M, and high in bR, late M, N and O. However, the precise nature of the interactions with the protein and the possible extent of out-of-plane distortions of the retinal that together determine the Schiff base pK and its position with respect to Asp85 in the lower proton channel and Asp96 in the upper proton channel are not yet obvious. (a) Structure of bR before isomerization of retinal. (b) Proton transfer from Sehiff base to Asp85. (c) Proton transfers from Asp96 to Schiff base and from Asp85 to the extracellular space. The timing of the transfer from Asp85 to the extracellular space is not yet clear. (d) Proton transfer from cytoplasmic surface to Asp96. different. Figure 20 shows only a minimal mechanism. The roles of Arg82, Tyr57 and Tyr185, as well as other serine and threonine residues and w a t e r molecules, have been omitted from the discussion, since they are not necessary to understand the basic mechanism. W a t e r molecules in particular, whether in the narrow, hydrophobic channel above the Schiff base or in the wider, hydrophilic channel below the Schiff base, are certain to play a role in
proton transfer as discussed by Nagle & Morowitz (1978}. Gating of the p a t h for the proton from the Schiff base first to the extracellular side, then from the cytoplasmic side might be mediated b y appropriate changes in hydrogen bonds without any change in the channel structure. Alternatively, it is possible t h a t the structural changes to form N with Asp96 u n p r o t o n a t e d cause the narrow hydrophobic chan-
Modal for Bacteriorhodopsin Structure nel above the Schiff base to open slightly so that solvation of the Asp96 carboxylate group is improved. In either case, it seems likely that the structural changes are relatively small. A number of measurements of charge movement across the membrane during the different steps of the photocycle have been made (Keszthelyi & Ormos, 1980; Trissl & G/~rtner, 1987; Butt et al., 1989; Otto et al., 1990). In most recent analyses, about 80 ~o of the charge movement occurs during the last part of the photocycle as M returns to bR (Butt et al., 1989). This is consistent with our finding that the upper proton channel is narrow and hydrophobic and is therefore the principal electrical barrier to movement of the proton across the membrane. Although the lower, extracellular proton channel is of equal length, its wider, hydrophilic character means it has space for water molecules and would present a lower barrier to movement of the proton. We expect that a more detailed understanding of the structure and mechanism of bR will be helped by higher-resolution analysis and refinement of the structure using the present model as a starting point. Further comprehensive analysis of the many different mutants is also critical with the eventual goal of a detailed dissection of the energetics of proton pumping.
(d) Refinement of the structure and study of structural changes Given a reasonable starting atomic model, such as in the present case, and diffraction amplitudes that extend beyond 3/k and up to 60 ° tilt (Ceska & Henderson, 1990), it should be possible to carry out a constrained refinement of the structure (Konnert & Hendrickson, 1980). This should give a more accurate structure, particularly in the vertical direction. Such refinement is in progress. I t should also be possible to investigate the structure of mutants, by the calculation of difference Fourier maps, if the mutants make good quality two-dimensional crystals. Glaeser et al. (1986) have demonstrated t h a t it is possible to trap an intermediate, which they believed to be M, at low temperature and collect good electron diffraction data. To get the greatest benefit from difference Fouriers, the errors in the diffraction data obtained must be less than the change due to the alteration of structure. In the case of bacteriorhodopsin electron diffraction data, we find the spread in amplitude measurements (overall mean value A A / A ) for typical 3 A diffraction patterns is about 8 to 1 0 ~ , suggesting that some improvements in accuracy are still required before difference Fouriers can show up some of the small'Changes that are expected to occur in the different photochemical intermediates. This is also a challenge for the future. The preliminary co-ordinates used in this model of bR are available on request from the authors and has been submitted to the Brookhaven data bank
927
together with the 2700 structure factors used to calculate the map. We thank EMBO for fellowships to R.H. and F.Z. and JEOL for supporting a visit by R.H. to use their 2000 SCM. K.H.D. was supported by grant number GM36884 from the US National Institutes of Health. We thank Jane Austin, Claudio Villa and staff of the photographic department at Cambridge for their help in producing the manuscript and illustrations. Finally, we thank our colleagues for their valuable comments on the manuscript and for their support and encouragement throughout this work. References Agard, D. A. (1983). J. Mol. Biol. 167, 849-852. Agard, D. A. & Stroud, R. M. (1982). Biophys. J. 37, 589-602. Argos, P., Rao, J. K. M. & Hargrave, P.A. (1982). Eur. J. Biochem. 128, 565-575. Baldwin, J. & Henderson, R. (1984). Ultramicroscopy, 14, 319-336. Baldwin, J. M., Henderson, R., Beckman, E. & Zemlin, F. (1988). J. Mol. Biol. 202, 585-591. Blanck, A. & Oesterhelt, D. (1987). EMBO J. 6, 265-273. Blanck, A., Oesterhelt, D., Ferrando, E., Schegk, E. S. & Lottspeich, F. (1989). EMBO J. 8, 3963-3971. Braiman, M. S., Mogi, T., Marti, T., Stern, L.J., Khorana, H. G. & Rothschild, K. J. (1988a). Biochemistry, 27, 8516-8520. Braiman, M. S., Mogi, T., Stern, L. J., Hackett, N. R., Chao, B. H., Khorana, H. G. & Rothschild, K. J. (1988b). Proteins: Struct. Funct. Genet. 3, 219-229. Bullough, P. & Henderson, R. (1987). Ultramicroscopy, 21, 223-230. Butt, H. J., Fendler, K., Bamburg, E., Tittor, J. & Oesterhelt, D. (1989). EMBO J. 8, 1657-1663. Ceska, T. A. & Henderson, R. (1990). J. Mol. Biol. In the press. Crepeau, R. H. & Fram, E. K. (i981). Ultramicrosco'py, 6, 7-18. Crowther, R. A. & Sleytr, U. B. (1977). J. Uitrastruct. Res. 58, 41-49. De Groot, H. J. M., Harbison, G. S., Herzfeld, J. & Griffin, R. G. (1989). Biochemistry, 28, 3346-3353. Dietrich, I., Fox, F., Knapek, E., Lefranc, G., Nachtrieb, K., Weyl, R. & Zerbst, H. (1977). Ultramicroscopy, 2, 241-249. Downing, K. H. (1988). Ultramicroscopy, 24, 387-398. Downing, K. H. & Glaeser, R. M. (1986). Ultramicroscopy, 20, 269-278. Dunn, R., McCoy, J., Simsek, M., Majumdar, A., Chang, S. H., Ra]Bhandary, U. L. & Khorana, H. G. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 6744-6748. Durra, R. J., Haekett, N. R., McCoy, J. M., Chao, B. H., Kimura, K. & Khorana, H. G. (1987). J. Biol. Chem. 262, 9246-9254. Earnest, T. N., Roepe, P., Braiman, M. S., Gillespie, J. & Rothschild, K. J. (1986). Biochemistry, 25, 7793-7798. Eisenstein, L., Lin, S.-L., Dollinger, G., Odashima, K., Termini, J., Konno, K., Ding, W.-D. & Nakanishi, K. (1987). J. Amer. Chem. Soc. 109, 6860-6862. Engelhard, M., Gerwert, K., Hess, B., Kreutz, W. & Siebert, F. (1985). Biochemistry, 24, 400-407. Engelman, D. M. & Zaccai, G. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 5894-5898.
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Edited by D. DeRosier