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Recent developments in electron crystallography of protein crystals
Recent developments in electron crystallography of protein crystals Friedrich Zemlin Fritz-Haber-lnstitut der Max-Planck-Gesellschaft, Berlin, Germany In recent years, electron crystallography has been a very successful means of determining the high-resolution structure of many proteins, such as bacteriorhodopsin, the light-harvesting complex, porins (PhoE and OmpF), protein extracted from the ootheca of the praying mantis, archaebacterial surface layers, acetylcholine receptor, Ca2+-ATPase and gp32*l crystals. This success was based on advanced low-dose cryoelectron microscopy and a highly sophisticated digital image-processing methodology which takes into account the radiation damage caused by electron exposure. Particularly successful has been the structure determination of the purple membrane where, by combining biochemical information with the electron-microscopical density maps, the amino acid chains of bacteriorhodopsin have been localized with high precision. Current Opinion in Structural Biology 1991, 1:1023-1029
Introduction This review focuses on the use of electron crystallography in the three-dimensional structure determination of two-dimensional protein crystals [1 ]. The electron microscope is used for measuring the amplitudes and phases of the Fourier coefficients from crystal lattices. The amplitudes are obtained from electron-diffraction patterns, the phases retrieved from the Fourier transforms of the images. Although X-ray crystallography has long been established as a research tool in structural biology, electron crystallography of proteins was for a long time hindered by certain intrinsic complications: radiation damage in the protein crystals; very low contrast of the structure; and, aberrations of the electron microscope. It took many years to overcome these difficulties. In quite a number of papers, these problems and their solutions are described. Unfortunately, the space constraints do not permit the discussion of all of these valuable contributions; instead, the reader is directed to the relevant reviews on each particular topic. Here, only those articles that describe high-resolution structure based on electron crystallography are discussed.
Sensitivity of protein crystals The high vacuum inside the electron microscope can itself damage protein crystals as a result of dehydration. This denaturation can more or less be avoided by embedding the specimens in amorphous ice [2] or neutral media such as glucose [3,4]. The characteristics of sample preparation and image recording for highresolution electron microscopy have recently been re-
viewed by Baumeister and Herrmann [5°]. The radiation damage sustained by crystals during exposure to electr6iS~ at different temperatures has been the focus of many studies; most investigated the fading of the electron-diffraction pattern that accompanies increasing electron dose. The published results vary considerably because of the many variables that influence the fading: the material of the specimen; specimen preparation, e.g. specimen embedding; the examined spatial frequency; the current density in the specimen plane; partial gaseous pressures around the specimen; and, specimen temperature. These parameters are difficult to control simultaneously. An insight into the complexity of the radiation damage in biological electron microscopy has been provided by Zeitler [6°]: Although the quoted findings differ remarkably, a trend emerges that cooling stabilizes the specimen. Hence, it is no surprise that most successful high-resolution structure research has been achieved at low specimen temperatures. In particular, the heliumcooled superconducting objective lens of Dietrich [7,8] has proved this point, although an essential feature of this objective lens, the reliable mechanical stability of the specimen stage, also contributes to the favourable results. Another procedure for retaining the high-resolution structure during imaging of the protein crystal is smallspot scanning, which substantially reduces beam-induced motions [9,10,11°]. Small-spot illumination, however, usually gives low coherence. In order to retain high coherence and thus optimize phase contrast, the use of a field-emission gun is advisable [11°]. The most important requirement for keeping radiation damage to a tolerable level is the use of low-dose imaging, in which exposure of the specimen to the electron beam before and during the image recording is greatly
(~) Current Biology Ltd ISSN 0959-440X
1023
1024 Biophysicalmethods
Fig. 1. One of the 7,~ thick slices through the three-dimensional map of bacteriorhodopsin with the atomic model superimposed. At this level, the retinal and most of the surrounding residues can be seen. minimized. The structure is subsequently retrieved from the noisy underexposed micrograph by image processing, particularly by correlation averaging of the multitude of imaged unit cells.
A protein crystal is a weak-phase object The aim of electron crystallography is, of course, the reconstruction of the native structure with high fidelity and high resolution. Two-dimensional protein crystals embedded in a thin layer of glucose or amorphous ice are weak-phase objects and thus are well suited to electron crystallography. For weak-phase objects, the greyvalue distribution in the micrograph is a direct representation of the local mass thickness, but is smeared out by a 'point-spread function'. Hence, direct representation does not necessarily lead to proportionality of the grey values and mass thickness in the original micrograph. But when the point-spread function is taken into account in the subsequent image processing, the reconstructed image can be interpreted. Image processing using weak-phase contrast theory has been described in detail [1,4,12,13..].
lated to this protein with the electron microscopical density map. The three-dimensional map of the structure has a resolution of 3.5A in a direction parallel to the membrane plane, but a resolution lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven cz-helices. These features are interpreted as the bulky aromatic side chains of phenyl~lanine, tyrosine and tryptophan residues. Another feature, and a very dense one, is the 13-ionone ring of the retinal chromophore. Using these bulky side chains as guide points and assuming that the smaller bulges indicate side chains of leucine, an amino acid that occurs frequently in a-helices, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built (Fig. 1). This model of bacteriorhodopsin has led to a better understanding of the biochemical operation of this molecule. A different crystallization of bacteriorhodopsin, the orthorhombic purple membrane, has been studied by electron cryomicroscopy [15o]. Because, in orthorhombic orientation, the phases of the structure factors in the projection relative to the twofold axis must be either 0* or 180", it offers an excellent reference point against which to determine the accuracy of high-resolution phases in electron crystallography.
Purple membrane Light-harvesting complex The electron crystallography of protein crystals was pioneered with the structure analysis of purple membrane [3,4,12,13oo,14]. Step-by-step, electron microscopy and image processing have been refined, resulting in an atomic model for bacteriorhodopsin. This model is obtained by combining all the biochemical knowledge re-
Kiihlbrandt and Wang [16o-] have succeeded in obtaining a high-resolution three-dimensional structure analysis of the plant light-harvesting complex by electron crystallography. This is the first t~ne that a eukaryotic membrane-protein structure has been elucidated us-
Recent developments in electron crystallography of protein crystals Zemlin "1025 ing this technique. The three-dimensional map of the complex with 6A resolution shows three membranespanning at-helices in the monomer and 15 chlorophyU molecules (Fig. 2). The double-layer distribution of chlorophyll molecules in the protein complex (equivalent to the two leaflets of the lipid bilayer) explains the fast energy transfer within the complex and to adjacent reaction centres. The prerequisite for this analysis was, of course, the successful crystallization of the protein; a detailed description of this is given in [17.].
Fig. 2. Side view of the light-harvesting complex II monomer from a central position in the lipid bitayer (indicated by the white lines) outside the trimer. The monomer contains three membranespanning 0c-helices (A, B and C) and 15 chlorophyll molecules (1-15). Chlorophylls 6 and 12 are hidden behind the helix B. Published with permission [16"].
3.5A resolution [19,20], although the models of PhoE and OmpF differ in fine detail. The biological function of porin PhoE has been described elsewhere [21]. Future electron microscopy of porins will be greatly influenced and helped by the recent successful high-resolution X-ray structure analysis of Rhodobacter capsulatus porin at 1.8~, performed by Weiss et al. [22].
Fig. 3. Three-dimensional map of PhoE porin at 6A resolution, looking down onto the plane of the membrane. PhoE porin consists of a trimeric cylindrical wall of B-sheet with most of the strands oriented about 35° from the membrane normal. The substructure within the cylinder extends over almost the width of the cylinder; this substructure determines the size and transport selectivity of the channel.
0c-Helical coiled-coil protein from the ootheca of the praying mantis Porin channels in outer membranes of bacteria A three-dimensional model of porin PhoE (with .,.6A resolution parallel to the membrane and --.8A resolution normal to the membrane) has been reconstructed by electron crystallography [18..]. Diffraction pattems and images of PhoE porin crystals, embedded in trehalose and maintained at about -120°C, were recorded from samples tilted between 0 ° and 60 ° using low-dose imaging techniques. The biochemical knowledge that this protein mostly consists of [3-sheets is in agreement with the structures of the cylindrical walls, which are obviously built up of [3-sheets (Fig. 3). This finding is also in good agreement with older models of porins PhoE and OmpF, which were derived from the two-dimensional maps with
The protein extracted from the ootheca (egg case) of the female praying mantis shows a coiled-coil supersecondary structure when analyzed by electron crystallography [23"]. The crystal of this protein is very highly ordered; its electron-diffraction pattern shows a resolution beyond 1.5A. Using a 'spot-scan' method of electron imaging, micrographs of unstained crystals were obtained with ~ 4.~. resolution. A projection map was calculated using electron-diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1A Qt-helical repeat. As the et-helical coiled-coil is a common super-secondary structure in fibrous proteins, these initial results appear very encouraging for further structure analysis of similar proteins by electron crystallography.
1026
Biophysicalmethods Surface layer of the archaebacterium Sulfolobus acidocaldarius
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The surface layer of the archaebacterium Sulfolobus ac/doca/dar/us has been investigated by exploring various image-analysis and image-processing strategies [24..]. Straightforward correlation-averaging correction for translational disorder only, and lattice unbending in conjunction with filtration in the Fourier domain gave equivalent results. A careful examination of cross-correlation functions as well as multivariate statistical analysis of individual unit cells revealed the existence of interpenetrating domains which exhibited intrinsic p3 symmetry, but which were rotated with respect to each other by 60* and separated by twin boundaries (Fig. 4). Non-discriminative averaging over twinned crystal patches generates a higher (p6) class of symmetry, emphasizing the importance of selective averaging. In addition to the biological results achieved in the structure analysis of this protein crystal, the methodology used has been extended greatly. In elucidating the correct crystal symmetry of this structure, the multivariate statistical analysis has proved to be a useful tool, and may find further use in electron crystallography. Developments in the image processing of crystals have also occurred. The &peak filtering technique predominantly used in early studies only works properly for ideal crystals. Now, by using cross-correlation averaging to extract the best parts of images, and by unbending the distorted crystal lattices, image processing can also be applied to real crystals, i.e. crystals with defects. Moreover, a deformed protein crystal often looks like an inaccurate array of unit cells; this can be corrected by 'mathematical recrystallization' which incorporates a way of modelling the unit cells by stretching or shortening, magnifying or demagnifying and rotating [25. ]. Multivariate statistical analysis is a further refinement of the methodology. The image processing of real crystals is approaching the methods used for single particles.
Acetylcholine receptor Toyoshima and Unwin [26.o] have elucidated the threedimensional structure of nicotinic acetyicholine receptor in Torpedo mamorata postsynaptic membranes by cryoelectron microscopy. The acetyicholine receptors were crystallized in the native lipids that formed tubular vesicles in which the molecules were arranged on a helical surface lattice. These vesicles were rapidly frozen and embedded in ice. Low-dose images with only 10 electrons per A2 imaged area were recorded at different defocus values and evaluated using the Fourier-Bessel theory for helical image reconstruction. The sophisticated ira-
Fig. 4.
(a) Twin boundary separating the major domains of the crystal. (b, c) Selective averages from the first row of unit cells on both sides along the twin boundary. They show the features of the p3 maps but with 60° rotational difference (separated by the boundary).
Recent developments in electron crystallography of protein crystals Zemlin age processing resulted in a three-dimensional map with 17Aresolution in all directions. The map revealed the organization of the subunits around the central ion pathway and in relation to the inner and outer leaflets of the lipid bilayer. The ion pathway itself could be resolved into three parts: two 20-25A wide entrance domains and a narrow pore spanning the lipid bilayer (Fig. 5). Tubular crystals are potentially ideal specimens for highresolution structure determination because they contain all views of a molecule over the tilt range of 0-90*. Thus, there is no 'missing cone' of information as in the case of a structure solved from a two-dimensional crystal.
Ca 2 + -ATPase For the structure determination of Ca2 + -ATPase, Stokes and Green [27-,28.-] used some quite different methods of investigation. The results complemented each other very well. The symmetry and molecular packing were investigated by electron microscopy of negatively stained crystals [27"]. By altering the detergent:lipid ratio, crystals of different size were produced, which adhered to supporting film in different orientations. In this way, micrographs of three different projections were obtained with the unit-ceU dimensions of 151 x 51 x 158 h and the three-dimensional space group C2 with an angle 13 very close to 90". These findings agree well with the crystal cell dimensions of 166 x 58 x 164h in hy-
drated unstained crystals that were previously obtained by X-ray powder diffraction. Micrographs from each of two principal projections were averaged to produce projection density maps. On the basis of these maps and a previously determined low-resolution structure of Ca2 +-ATPase, a packing diagram for these three-dimensional crystals was presented and the major intermolecular contacts were described. In a second paper, the authors studied unstained frozenhydrated thin crystals of Ca2 +-ATPase by electron crystaUography [28.-]. The resulting electron-diffraction patterns extended to 4.1 ~, resolution and the images contained phase data up to 6A resolution. By combining Fourier amplitudes from electron-diffraction patterns with phases from the images, a density map has been calculated in projection. Comparison of this map from unstained crystals with the previously determined map from negatively stained crystals [27"] reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and the packing of the molecules in the crystal, the authors proposed a specific arrangement for the 10 cx-helices that span the bilayer.
Analysis of thin g p 3 2 * l crystals The DNA-helix-destabilizing protein gp32 from T4 bacteriophage is a non-specific DNA-binding protein which
90"
Fig. 5. Cross-section through the acetyl-
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choline receptor tube, reconstructed from cryo-images. Individual receptors are intersected at different levels; only the one marked R is cut through the centre, thus showing the profile of the ion pathway. Note that the outer (0) and inner (I) leaflets of the lipid bilayer are also resolved.
1027
1028 Biophysicalmethods contains a zinc finger. An X-ray analysis of the structure is not yet available. Gp32 * I, a proteolytically cleaved protein derived from gp32 that retains its DNA-binding properties, can be suitably crystallized for electron crystallogt¢aphic analysis. The crystal forms with varying unit-cell dimensions. Grant et a t [29 o] used crystallographic image analysis to separate image areas corresponding to different thicknesses. A three-dimensional structure from a single layer of crystalline area was reconstructed from 110 tilted images and three domains of the protein were interpreted. This protein crystal is also a good candidate for higher-resolution structure determination because it can diffract beyond 3.5 A, resolution.
BAUMEISTERW, HERRMANN KH: High-resolution Electron Microscopy in Biology: Sample Preparation, Image Recording and Processing. In Biophysical Electron MtZroscopy edited by Hawkes P, Valdre U [book]. London: Academic Press, 1990, pp 108-131. A mini-review about the major problems that attend the sample preparation, image recording and processing of biological specimens for electron microscopy.
7.
DIETRICH1, FOX F, LEFRANCG, NACHTRIEBK, WEYL R, ZERBST H: Improvements in Electron Microscopy by Application of Superconductivity. Ultramicroscopy 1977, 2:241-249.
Conclusion
8.
LEFRANCG, KNAPEK K, DIETRICH I: Superconducting Lens Design. Ultramicroscopy 1982, 10:111-124.
The major breakthrough in electron crystallography of proteins has been the combination of low-dose cryoelectron microscopy with powerful computer image processing. One significant problem that remains to be solved is the crystallization of proteins in two-dimensional crystals. For electron crystallography, minute crystals of ,-, 1 I,tm diameter are sufficient. This can be regarded as an advantage of electron crystallography over X-my crystallography. Baumeister and Herrmann [5"] hit the nail on the head in summing up the status of electron crystallography thus: "Electron crystallography is on the brink of establishing itself as a technique capable of analysing macromolecular structure with a precision that had hitherto been considered as exclusive to the realm of X-ray crystallography".
9.
HENDERSONR, GLAESERRM: Quantitative Analysis of Image Contrast in Electron Micrographs of Beam-sensitive Crystals. U l t r a m i c ~ 1985, 16:139-150.
10.
BULLOUGHP, HENDERSONR: Use of Spot-scan Procedure for Recording Low-dose Micrographs of Beam-sensitive Specimens. Ultramicraccopy 1987, 21:223-230.
5. .
ZEITLERE: Radiation Damage in Biological Electron Microscopy. In Biophysical Electron Microscopy edited by Hawkes P, Valdre U [book]. London: Academic Press, 1990, pp 288-308. A short description of the main effects of radiation damage and effective counter measures. 6. •
11. DOWNINGK: Spot-scan Imaging in Transmission Electron Mi• croscopy. Science 1991, 251:53-59. Taking various specimens as examples, this review describes the benefits of the spot-scan imaging method. 12.
HENDERSONR, BALDWINJM, DOWNING KH, LEPAULTJ, ZEMLINF: Structure o f ~ Membrane from Halobacterium halt> biur~ Recording, Measurement and Evaluation of Electron Micrographs at 3.5A Resolution. Ultramicroscopy 1986, 19:147-178.
13.
HENDERSONR, BALDWINJM, CESKATA, ZEMLIN F, BECKMANNE, DOWNINGKH: Model for the Structure of Bacteriorhodopsin Based on High-resolution Electron Cryo-microscopy. J Mo/ Biol 1990, 213:899-929. The very first atomic model of a biological molecule based on electron crystallography is described. A very clear introduction to the methodology is also given. ••
Acknowledgements The author is grateful to E Zeitler for encouraging this paper, and thanks P Dube, M van Heel and R Henderson for helpful discussions.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest ** of outstanding interest
14.
BALDWINJM, HENDERSON R, BACKMANNE, ZEMLINF: Image of Purple Membrane at 2.8h Resolution Obtained by Cryoelectron Microscopy. J Mol Biol 1988, 202:585--591.
15. .
BULLOUGH PA, HENDERSON R: Phase Accuracy in Highresolution Electron Microscopy of Trigonal and Orthogonal Purple Membrane. BiophysJ 1990, 58:705-711. Electron crystallography of orthorhombic purple membrane yields a projection structure with 3.9A resolution. KUHLBRANDTW, WANG DN: Three-dimensional Structure of Plant Light-harvesting Complex Determined by Electron Crystallography. Nature 1991, 350:130-134. The three-dimensional structure of the light-harvesting complex, the major antenna for solar energy in plant photosynthesis, is described. It is the first euka~otic membrane-protein structure to be elucidated up to 6A by electron crystallography. 16. .•
1.
AMOS LA, HENDERSON R, UNWIN PNT: Three-dimensional Structure Determination by Electron Microscopy of Twodimensional Crystals. Prog Biophys Mol Biol 1982, 39:188231.
2.
DUBOCHETJ, ADRIAN M, CHANG JJ, HOMO JC, LEPAULT J, McDOWALLAW, SCHULTZP: Cryo-eleetfon Microscopy of Vitrified Specimens. Q Rev Biophys 1988, 21:129-228.
WANGDN, KOHLBRANDTW: High-resolution Electron Crystallography of Light-harvesting Chlorophyll a/b-Protein Complex in Three Different Media. J Mol Biol 1991, 217:691~99. The crystallization of the light-harvesting complex is described in detail.
3.
HENDERSONR, UNWlN PNT: Three-dimensional Model of Purple Membrane Obtained by Electron Microscopy. Nature 1975, 257:28-32.
18. •.
4.
UNWINPNT, HENDERSON R: Molecular Structure Determination by Electron Microscopy o f Unstained Crystalline Specimens. J Mol Biol 1975, 94:425-440.
17. .
JAP BK, WALIAN PJ, GEHPdNG K: Structural Architecture o f an Outer Membrane Channel as Determined by Electron Crystallography. Nature 1991, 350:167-170. P o r i n - a family of membrane channels found in Gram-negative bacteria-elucidated up to 6A by electron crystallography shows a 13-sheet barrel structure as predicted earlier using spectroscopic methods.
Recent d e v e l o p m e n t s in e l e c t r o n c r y s t a l l o g r a p h y of protein crystals Zemlin 19.
JAP BK, DOWNINGKH, WALIANPJ: Structure of PhoE Porin in Projection at 3.5 A Resolution. J Struct B~/1990, 103:57-63.
20.
SASSHJ, BOLDTG, BECKMANNE, ZEMLINF, VAN HEELM, ZEITLER E, ROSENBUSCHJP, DORSET DI, MASSALSKIA: Densely Packed IS-Structure at the Protein-Lipid Interface of Porin is Revealed by I-limb-resolution Cryo-electron Microscopy. J Mol B/o/1989, 209:171-175.
21.
JAP BK: Molecular Design of PhoE Porin and its Functional Consequences. J Mol Biol 1989, 205:407-419.
22.
WEISS MS, KREUSCH A, SCHIL'IZ E, NESTEL U, WELTE W, WECKESSER J, SCHULZ GE: The Structure of Porin from Rhodobacter capsulatus at 1.8A Resolution. FEBS Lett 1991, 280:379-382.
23. •
BULLOUGHPA, "DM~H P& Hi,,h-resolution Spot-scan Electron Microscopy of an 0t-Helical Coiled-coil Protein. J Mol Biol 1990, 215:161-173. The s-helical coiled-coil protein extracted from the ootheca of the praying mantis is investigated. A projection map is calculated to 43[ resolution and reveals a 5.1A or-helical relx~t.
24. **
LEMBCKEG, DORR R, HEGERLR, BAUMEISTERW: Image Anal}'sis and Processing of an Imperfect Two-dimensional Crystal: the Surface Layer of the Archaebacterium Sulfolobus acidocaldar~us Re-investigated. J Mz~rc~cc~ 1991, 161:263-278. Employing the mult~wariatestatistical analysis, the correct symmetry of the structure is determined. This work presents an example of careful image analysis of native crystalline smctures where crystal defects are commonplace. 25. •
DORR17, HEGERL R, VOLKER S, SANTARIUSU, BAUMEISTERW: Three-dimensional Reconstruction of the Surface Protein
of P y r ~ t c t t u m brockii Comparing Two Image Processing Strategies. J Smact Biol 1991, 106:181-190. 26. **
TOYOSHIMAC, UNWIN PNT: Three-dimensional Structure of the Acetylcholine Receptor by Cryoelectron Microscopy and Helical Image Reconstruction. J Cell Bicd 1990, 111:2623-2635. Using the techniques mentioned, the three-dimensionaJ stucmre of nicotinic acetlycholine receptor is resolved to 17~ which clearly shows the intra-membrane ion Wathwayof the channel. 27. .
STOKESDL, GREEN NM: Three-dimensional Crystals of CaATPase from Sarcoplasmic reticulum: Symmetry and Molecular Paddng. J B/q0hys 1990, 57:1-4.
28. .•
STOKESDL, GREEN NM: Structure of CaATPase: Electron Microscopy of Frozen-hydrated Crystals at 6A Resolution in Projection. J MM B:~ 1990, 213:529-538. Combining the electron-density maps of stained and unstained crystals, a model of Ca2+ -ATPase is described in which membranous and extramembranous domains can be distinguished. GRANTRA, SCHMID ME, CHIU W: Analysis of Symmetry and Three-dimensional Reconstruction of Thin gp32"I crystals. J Mol Biol 1991, 217:551-562. Thin multilayered crystals of gp32*l are investigated by negative stain electron microscopy and image processing. The four types of projections produced are systematically analyzed, resulting in a three-dimensional reconstruction of the molecule with ~ 18./[ resolution in all directions (some data out to 15A). 29. •
F Zemlin, Fritz-Haber-lnstitut der Max-Planck-Gesellschaft, Faradayweg 4~6, D-1000 Berlin 33, Germany.
1029
Introduction This review focuses on the use of electron crystallography in the three-dimensional structure determination of two-dimensional protein crystals [1 ]. The electron microscope is used for measuring the amplitudes and phases of the Fourier coefficients from crystal lattices. The amplitudes are obtained from electron-diffraction patterns, the phases retrieved from the Fourier transforms of the images. Although X-ray crystallography has long been established as a research tool in structural biology, electron crystallography of proteins was for a long time hindered by certain intrinsic complications: radiation damage in the protein crystals; very low contrast of the structure; and, aberrations of the electron microscope. It took many years to overcome these difficulties. In quite a number of papers, these problems and their solutions are described. Unfortunately, the space constraints do not permit the discussion of all of these valuable contributions; instead, the reader is directed to the relevant reviews on each particular topic. Here, only those articles that describe high-resolution structure based on electron crystallography are discussed.
Sensitivity of protein crystals The high vacuum inside the electron microscope can itself damage protein crystals as a result of dehydration. This denaturation can more or less be avoided by embedding the specimens in amorphous ice [2] or neutral media such as glucose [3,4]. The characteristics of sample preparation and image recording for highresolution electron microscopy have recently been re-
viewed by Baumeister and Herrmann [5°]. The radiation damage sustained by crystals during exposure to electr6iS~ at different temperatures has been the focus of many studies; most investigated the fading of the electron-diffraction pattern that accompanies increasing electron dose. The published results vary considerably because of the many variables that influence the fading: the material of the specimen; specimen preparation, e.g. specimen embedding; the examined spatial frequency; the current density in the specimen plane; partial gaseous pressures around the specimen; and, specimen temperature. These parameters are difficult to control simultaneously. An insight into the complexity of the radiation damage in biological electron microscopy has been provided by Zeitler [6°]: Although the quoted findings differ remarkably, a trend emerges that cooling stabilizes the specimen. Hence, it is no surprise that most successful high-resolution structure research has been achieved at low specimen temperatures. In particular, the heliumcooled superconducting objective lens of Dietrich [7,8] has proved this point, although an essential feature of this objective lens, the reliable mechanical stability of the specimen stage, also contributes to the favourable results. Another procedure for retaining the high-resolution structure during imaging of the protein crystal is smallspot scanning, which substantially reduces beam-induced motions [9,10,11°]. Small-spot illumination, however, usually gives low coherence. In order to retain high coherence and thus optimize phase contrast, the use of a field-emission gun is advisable [11°]. The most important requirement for keeping radiation damage to a tolerable level is the use of low-dose imaging, in which exposure of the specimen to the electron beam before and during the image recording is greatly
(~) Current Biology Ltd ISSN 0959-440X
1023
1024 Biophysicalmethods
Fig. 1. One of the 7,~ thick slices through the three-dimensional map of bacteriorhodopsin with the atomic model superimposed. At this level, the retinal and most of the surrounding residues can be seen. minimized. The structure is subsequently retrieved from the noisy underexposed micrograph by image processing, particularly by correlation averaging of the multitude of imaged unit cells.
A protein crystal is a weak-phase object The aim of electron crystallography is, of course, the reconstruction of the native structure with high fidelity and high resolution. Two-dimensional protein crystals embedded in a thin layer of glucose or amorphous ice are weak-phase objects and thus are well suited to electron crystallography. For weak-phase objects, the greyvalue distribution in the micrograph is a direct representation of the local mass thickness, but is smeared out by a 'point-spread function'. Hence, direct representation does not necessarily lead to proportionality of the grey values and mass thickness in the original micrograph. But when the point-spread function is taken into account in the subsequent image processing, the reconstructed image can be interpreted. Image processing using weak-phase contrast theory has been described in detail [1,4,12,13..].
lated to this protein with the electron microscopical density map. The three-dimensional map of the structure has a resolution of 3.5A in a direction parallel to the membrane plane, but a resolution lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven cz-helices. These features are interpreted as the bulky aromatic side chains of phenyl~lanine, tyrosine and tryptophan residues. Another feature, and a very dense one, is the 13-ionone ring of the retinal chromophore. Using these bulky side chains as guide points and assuming that the smaller bulges indicate side chains of leucine, an amino acid that occurs frequently in a-helices, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built (Fig. 1). This model of bacteriorhodopsin has led to a better understanding of the biochemical operation of this molecule. A different crystallization of bacteriorhodopsin, the orthorhombic purple membrane, has been studied by electron cryomicroscopy [15o]. Because, in orthorhombic orientation, the phases of the structure factors in the projection relative to the twofold axis must be either 0* or 180", it offers an excellent reference point against which to determine the accuracy of high-resolution phases in electron crystallography.
Purple membrane Light-harvesting complex The electron crystallography of protein crystals was pioneered with the structure analysis of purple membrane [3,4,12,13oo,14]. Step-by-step, electron microscopy and image processing have been refined, resulting in an atomic model for bacteriorhodopsin. This model is obtained by combining all the biochemical knowledge re-
Kiihlbrandt and Wang [16o-] have succeeded in obtaining a high-resolution three-dimensional structure analysis of the plant light-harvesting complex by electron crystallography. This is the first t~ne that a eukaryotic membrane-protein structure has been elucidated us-
Recent developments in electron crystallography of protein crystals Zemlin "1025 ing this technique. The three-dimensional map of the complex with 6A resolution shows three membranespanning at-helices in the monomer and 15 chlorophyU molecules (Fig. 2). The double-layer distribution of chlorophyll molecules in the protein complex (equivalent to the two leaflets of the lipid bilayer) explains the fast energy transfer within the complex and to adjacent reaction centres. The prerequisite for this analysis was, of course, the successful crystallization of the protein; a detailed description of this is given in [17.].
Fig. 2. Side view of the light-harvesting complex II monomer from a central position in the lipid bitayer (indicated by the white lines) outside the trimer. The monomer contains three membranespanning 0c-helices (A, B and C) and 15 chlorophyll molecules (1-15). Chlorophylls 6 and 12 are hidden behind the helix B. Published with permission [16"].
3.5A resolution [19,20], although the models of PhoE and OmpF differ in fine detail. The biological function of porin PhoE has been described elsewhere [21]. Future electron microscopy of porins will be greatly influenced and helped by the recent successful high-resolution X-ray structure analysis of Rhodobacter capsulatus porin at 1.8~, performed by Weiss et al. [22].
Fig. 3. Three-dimensional map of PhoE porin at 6A resolution, looking down onto the plane of the membrane. PhoE porin consists of a trimeric cylindrical wall of B-sheet with most of the strands oriented about 35° from the membrane normal. The substructure within the cylinder extends over almost the width of the cylinder; this substructure determines the size and transport selectivity of the channel.
0c-Helical coiled-coil protein from the ootheca of the praying mantis Porin channels in outer membranes of bacteria A three-dimensional model of porin PhoE (with .,.6A resolution parallel to the membrane and --.8A resolution normal to the membrane) has been reconstructed by electron crystallography [18..]. Diffraction pattems and images of PhoE porin crystals, embedded in trehalose and maintained at about -120°C, were recorded from samples tilted between 0 ° and 60 ° using low-dose imaging techniques. The biochemical knowledge that this protein mostly consists of [3-sheets is in agreement with the structures of the cylindrical walls, which are obviously built up of [3-sheets (Fig. 3). This finding is also in good agreement with older models of porins PhoE and OmpF, which were derived from the two-dimensional maps with
The protein extracted from the ootheca (egg case) of the female praying mantis shows a coiled-coil supersecondary structure when analyzed by electron crystallography [23"]. The crystal of this protein is very highly ordered; its electron-diffraction pattern shows a resolution beyond 1.5A. Using a 'spot-scan' method of electron imaging, micrographs of unstained crystals were obtained with ~ 4.~. resolution. A projection map was calculated using electron-diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1A Qt-helical repeat. As the et-helical coiled-coil is a common super-secondary structure in fibrous proteins, these initial results appear very encouraging for further structure analysis of similar proteins by electron crystallography.
1026
Biophysicalmethods Surface layer of the archaebacterium Sulfolobus acidocaldarius
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The surface layer of the archaebacterium Sulfolobus ac/doca/dar/us has been investigated by exploring various image-analysis and image-processing strategies [24..]. Straightforward correlation-averaging correction for translational disorder only, and lattice unbending in conjunction with filtration in the Fourier domain gave equivalent results. A careful examination of cross-correlation functions as well as multivariate statistical analysis of individual unit cells revealed the existence of interpenetrating domains which exhibited intrinsic p3 symmetry, but which were rotated with respect to each other by 60* and separated by twin boundaries (Fig. 4). Non-discriminative averaging over twinned crystal patches generates a higher (p6) class of symmetry, emphasizing the importance of selective averaging. In addition to the biological results achieved in the structure analysis of this protein crystal, the methodology used has been extended greatly. In elucidating the correct crystal symmetry of this structure, the multivariate statistical analysis has proved to be a useful tool, and may find further use in electron crystallography. Developments in the image processing of crystals have also occurred. The &peak filtering technique predominantly used in early studies only works properly for ideal crystals. Now, by using cross-correlation averaging to extract the best parts of images, and by unbending the distorted crystal lattices, image processing can also be applied to real crystals, i.e. crystals with defects. Moreover, a deformed protein crystal often looks like an inaccurate array of unit cells; this can be corrected by 'mathematical recrystallization' which incorporates a way of modelling the unit cells by stretching or shortening, magnifying or demagnifying and rotating [25. ]. Multivariate statistical analysis is a further refinement of the methodology. The image processing of real crystals is approaching the methods used for single particles.
Acetylcholine receptor Toyoshima and Unwin [26.o] have elucidated the threedimensional structure of nicotinic acetyicholine receptor in Torpedo mamorata postsynaptic membranes by cryoelectron microscopy. The acetyicholine receptors were crystallized in the native lipids that formed tubular vesicles in which the molecules were arranged on a helical surface lattice. These vesicles were rapidly frozen and embedded in ice. Low-dose images with only 10 electrons per A2 imaged area were recorded at different defocus values and evaluated using the Fourier-Bessel theory for helical image reconstruction. The sophisticated ira-
Fig. 4.
(a) Twin boundary separating the major domains of the crystal. (b, c) Selective averages from the first row of unit cells on both sides along the twin boundary. They show the features of the p3 maps but with 60° rotational difference (separated by the boundary).
Recent developments in electron crystallography of protein crystals Zemlin age processing resulted in a three-dimensional map with 17Aresolution in all directions. The map revealed the organization of the subunits around the central ion pathway and in relation to the inner and outer leaflets of the lipid bilayer. The ion pathway itself could be resolved into three parts: two 20-25A wide entrance domains and a narrow pore spanning the lipid bilayer (Fig. 5). Tubular crystals are potentially ideal specimens for highresolution structure determination because they contain all views of a molecule over the tilt range of 0-90*. Thus, there is no 'missing cone' of information as in the case of a structure solved from a two-dimensional crystal.
Ca 2 + -ATPase For the structure determination of Ca2 + -ATPase, Stokes and Green [27-,28.-] used some quite different methods of investigation. The results complemented each other very well. The symmetry and molecular packing were investigated by electron microscopy of negatively stained crystals [27"]. By altering the detergent:lipid ratio, crystals of different size were produced, which adhered to supporting film in different orientations. In this way, micrographs of three different projections were obtained with the unit-ceU dimensions of 151 x 51 x 158 h and the three-dimensional space group C2 with an angle 13 very close to 90". These findings agree well with the crystal cell dimensions of 166 x 58 x 164h in hy-
drated unstained crystals that were previously obtained by X-ray powder diffraction. Micrographs from each of two principal projections were averaged to produce projection density maps. On the basis of these maps and a previously determined low-resolution structure of Ca2 +-ATPase, a packing diagram for these three-dimensional crystals was presented and the major intermolecular contacts were described. In a second paper, the authors studied unstained frozenhydrated thin crystals of Ca2 +-ATPase by electron crystaUography [28.-]. The resulting electron-diffraction patterns extended to 4.1 ~, resolution and the images contained phase data up to 6A resolution. By combining Fourier amplitudes from electron-diffraction patterns with phases from the images, a density map has been calculated in projection. Comparison of this map from unstained crystals with the previously determined map from negatively stained crystals [27"] reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and the packing of the molecules in the crystal, the authors proposed a specific arrangement for the 10 cx-helices that span the bilayer.
Analysis of thin g p 3 2 * l crystals The DNA-helix-destabilizing protein gp32 from T4 bacteriophage is a non-specific DNA-binding protein which
90"
Fig. 5. Cross-section through the acetyl-
radius
03s0A
choline receptor tube, reconstructed from cryo-images. Individual receptors are intersected at different levels; only the one marked R is cut through the centre, thus showing the profile of the ion pathway. Note that the outer (0) and inner (I) leaflets of the lipid bilayer are also resolved.
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1028 Biophysicalmethods contains a zinc finger. An X-ray analysis of the structure is not yet available. Gp32 * I, a proteolytically cleaved protein derived from gp32 that retains its DNA-binding properties, can be suitably crystallized for electron crystallogt¢aphic analysis. The crystal forms with varying unit-cell dimensions. Grant et a t [29 o] used crystallographic image analysis to separate image areas corresponding to different thicknesses. A three-dimensional structure from a single layer of crystalline area was reconstructed from 110 tilted images and three domains of the protein were interpreted. This protein crystal is also a good candidate for higher-resolution structure determination because it can diffract beyond 3.5 A, resolution.
BAUMEISTERW, HERRMANN KH: High-resolution Electron Microscopy in Biology: Sample Preparation, Image Recording and Processing. In Biophysical Electron MtZroscopy edited by Hawkes P, Valdre U [book]. London: Academic Press, 1990, pp 108-131. A mini-review about the major problems that attend the sample preparation, image recording and processing of biological specimens for electron microscopy.
7.
DIETRICH1, FOX F, LEFRANCG, NACHTRIEBK, WEYL R, ZERBST H: Improvements in Electron Microscopy by Application of Superconductivity. Ultramicroscopy 1977, 2:241-249.
Conclusion
8.
LEFRANCG, KNAPEK K, DIETRICH I: Superconducting Lens Design. Ultramicroscopy 1982, 10:111-124.
The major breakthrough in electron crystallography of proteins has been the combination of low-dose cryoelectron microscopy with powerful computer image processing. One significant problem that remains to be solved is the crystallization of proteins in two-dimensional crystals. For electron crystallography, minute crystals of ,-, 1 I,tm diameter are sufficient. This can be regarded as an advantage of electron crystallography over X-my crystallography. Baumeister and Herrmann [5"] hit the nail on the head in summing up the status of electron crystallography thus: "Electron crystallography is on the brink of establishing itself as a technique capable of analysing macromolecular structure with a precision that had hitherto been considered as exclusive to the realm of X-ray crystallography".
9.
HENDERSONR, GLAESERRM: Quantitative Analysis of Image Contrast in Electron Micrographs of Beam-sensitive Crystals. U l t r a m i c ~ 1985, 16:139-150.
10.
BULLOUGHP, HENDERSONR: Use of Spot-scan Procedure for Recording Low-dose Micrographs of Beam-sensitive Specimens. Ultramicraccopy 1987, 21:223-230.
5. .
ZEITLERE: Radiation Damage in Biological Electron Microscopy. In Biophysical Electron Microscopy edited by Hawkes P, Valdre U [book]. London: Academic Press, 1990, pp 288-308. A short description of the main effects of radiation damage and effective counter measures. 6. •
11. DOWNINGK: Spot-scan Imaging in Transmission Electron Mi• croscopy. Science 1991, 251:53-59. Taking various specimens as examples, this review describes the benefits of the spot-scan imaging method. 12.
HENDERSONR, BALDWINJM, DOWNING KH, LEPAULTJ, ZEMLINF: Structure o f ~ Membrane from Halobacterium halt> biur~ Recording, Measurement and Evaluation of Electron Micrographs at 3.5A Resolution. Ultramicroscopy 1986, 19:147-178.
13.
HENDERSONR, BALDWINJM, CESKATA, ZEMLIN F, BECKMANNE, DOWNINGKH: Model for the Structure of Bacteriorhodopsin Based on High-resolution Electron Cryo-microscopy. J Mo/ Biol 1990, 213:899-929. The very first atomic model of a biological molecule based on electron crystallography is described. A very clear introduction to the methodology is also given. ••
Acknowledgements The author is grateful to E Zeitler for encouraging this paper, and thanks P Dube, M van Heel and R Henderson for helpful discussions.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest ** of outstanding interest
14.
BALDWINJM, HENDERSON R, BACKMANNE, ZEMLINF: Image of Purple Membrane at 2.8h Resolution Obtained by Cryoelectron Microscopy. J Mol Biol 1988, 202:585--591.
15. .
BULLOUGH PA, HENDERSON R: Phase Accuracy in Highresolution Electron Microscopy of Trigonal and Orthogonal Purple Membrane. BiophysJ 1990, 58:705-711. Electron crystallography of orthorhombic purple membrane yields a projection structure with 3.9A resolution. KUHLBRANDTW, WANG DN: Three-dimensional Structure of Plant Light-harvesting Complex Determined by Electron Crystallography. Nature 1991, 350:130-134. The three-dimensional structure of the light-harvesting complex, the major antenna for solar energy in plant photosynthesis, is described. It is the first euka~otic membrane-protein structure to be elucidated up to 6A by electron crystallography. 16. .•
1.
AMOS LA, HENDERSON R, UNWIN PNT: Three-dimensional Structure Determination by Electron Microscopy of Twodimensional Crystals. Prog Biophys Mol Biol 1982, 39:188231.
2.
DUBOCHETJ, ADRIAN M, CHANG JJ, HOMO JC, LEPAULT J, McDOWALLAW, SCHULTZP: Cryo-eleetfon Microscopy of Vitrified Specimens. Q Rev Biophys 1988, 21:129-228.
WANGDN, KOHLBRANDTW: High-resolution Electron Crystallography of Light-harvesting Chlorophyll a/b-Protein Complex in Three Different Media. J Mol Biol 1991, 217:691~99. The crystallization of the light-harvesting complex is described in detail.
3.
HENDERSONR, UNWlN PNT: Three-dimensional Model of Purple Membrane Obtained by Electron Microscopy. Nature 1975, 257:28-32.
18. •.
4.
UNWINPNT, HENDERSON R: Molecular Structure Determination by Electron Microscopy o f Unstained Crystalline Specimens. J Mol Biol 1975, 94:425-440.
17. .
JAP BK, WALIAN PJ, GEHPdNG K: Structural Architecture o f an Outer Membrane Channel as Determined by Electron Crystallography. Nature 1991, 350:167-170. P o r i n - a family of membrane channels found in Gram-negative bacteria-elucidated up to 6A by electron crystallography shows a 13-sheet barrel structure as predicted earlier using spectroscopic methods.
Recent d e v e l o p m e n t s in e l e c t r o n c r y s t a l l o g r a p h y of protein crystals Zemlin 19.
JAP BK, DOWNINGKH, WALIANPJ: Structure of PhoE Porin in Projection at 3.5 A Resolution. J Struct B~/1990, 103:57-63.
20.
SASSHJ, BOLDTG, BECKMANNE, ZEMLINF, VAN HEELM, ZEITLER E, ROSENBUSCHJP, DORSET DI, MASSALSKIA: Densely Packed IS-Structure at the Protein-Lipid Interface of Porin is Revealed by I-limb-resolution Cryo-electron Microscopy. J Mol B/o/1989, 209:171-175.
21.
JAP BK: Molecular Design of PhoE Porin and its Functional Consequences. J Mol Biol 1989, 205:407-419.
22.
WEISS MS, KREUSCH A, SCHIL'IZ E, NESTEL U, WELTE W, WECKESSER J, SCHULZ GE: The Structure of Porin from Rhodobacter capsulatus at 1.8A Resolution. FEBS Lett 1991, 280:379-382.
23. •
BULLOUGHPA, "DM~H P& Hi,,h-resolution Spot-scan Electron Microscopy of an 0t-Helical Coiled-coil Protein. J Mol Biol 1990, 215:161-173. The s-helical coiled-coil protein extracted from the ootheca of the praying mantis is investigated. A projection map is calculated to 43[ resolution and reveals a 5.1A or-helical relx~t.
24. **
LEMBCKEG, DORR R, HEGERLR, BAUMEISTERW: Image Anal}'sis and Processing of an Imperfect Two-dimensional Crystal: the Surface Layer of the Archaebacterium Sulfolobus acidocaldar~us Re-investigated. J Mz~rc~cc~ 1991, 161:263-278. Employing the mult~wariatestatistical analysis, the correct symmetry of the structure is determined. This work presents an example of careful image analysis of native crystalline smctures where crystal defects are commonplace. 25. •
DORR17, HEGERL R, VOLKER S, SANTARIUSU, BAUMEISTERW: Three-dimensional Reconstruction of the Surface Protein
of P y r ~ t c t t u m brockii Comparing Two Image Processing Strategies. J Smact Biol 1991, 106:181-190. 26. **
TOYOSHIMAC, UNWIN PNT: Three-dimensional Structure of the Acetylcholine Receptor by Cryoelectron Microscopy and Helical Image Reconstruction. J Cell Bicd 1990, 111:2623-2635. Using the techniques mentioned, the three-dimensionaJ stucmre of nicotinic acetlycholine receptor is resolved to 17~ which clearly shows the intra-membrane ion Wathwayof the channel. 27. .
STOKESDL, GREEN NM: Three-dimensional Crystals of CaATPase from Sarcoplasmic reticulum: Symmetry and Molecular Paddng. J B/q0hys 1990, 57:1-4.
28. .•
STOKESDL, GREEN NM: Structure of CaATPase: Electron Microscopy of Frozen-hydrated Crystals at 6A Resolution in Projection. J MM B:~ 1990, 213:529-538. Combining the electron-density maps of stained and unstained crystals, a model of Ca2+ -ATPase is described in which membranous and extramembranous domains can be distinguished. GRANTRA, SCHMID ME, CHIU W: Analysis of Symmetry and Three-dimensional Reconstruction of Thin gp32"I crystals. J Mol Biol 1991, 217:551-562. Thin multilayered crystals of gp32*l are investigated by negative stain electron microscopy and image processing. The four types of projections produced are systematically analyzed, resulting in a three-dimensional reconstruction of the molecule with ~ 18./[ resolution in all directions (some data out to 15A). 29. •
F Zemlin, Fritz-Haber-lnstitut der Max-Planck-Gesellschaft, Faradayweg 4~6, D-1000 Berlin 33, Germany.
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