- Email: [email protected]
High-resolution electron microscopy of biological specimens in cubic ice
ultramicroscopy ELSEVIER
Ultramicroscopy 55 (1994) 141-153
High-resolution electron microscopy of biological specimens in cubic ice Marek Cyrklaff, Werner Kiihlbrandt European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Received 1 December 1993; in final form 4 May 1994
Abstract
Images of two biological test specimens, catalase and TMV, were recorded in cubic ice, prepared by controlled devitrification at -130°C and -75°C or in vitrified buffer. Cubic ice provides a rigid support for biological specimens which is stable under the electron beam to within about 1 ,~, as shown by images of the ice lattice. Neither catalase nor TMV were disrupted by the crystallization of vitrified water. Electron diffraction patterns of highly oriented rafts of TMV extending to 2.3 ,& resolution were used to judge the quality of TMV images. Structural features at 5.75 ,& (TMV) and 5.4 ,& (catalase) were observed directly by optical diffraction. The reproduction of high-resolution details of TMV was better in cubic ice, and the success rate for recording good images was higher than in vitrified medium.
1. Introduction
The structure of biological macromolecules and macromolecular assemblies can be determined by electron microscopy at atomic or near-atomic resolution, provided that it is possible to record images in which high-resolution features of the object are faithfully reproduced. This requires (a) a n instrument which is capable of recording atomic resolution images of biological specimens and (b) that the object is preserved in its native, hydrated form. So far, images with resolutions between 3 and 4 A have been recorded primarily of two-dimensional (2D) and thin three-dimensional (3D) protein crystals. 2D crystals imaged at high resolution include bacteriorhodopsin [1,2], the bacterial porins PhoE [3], O m p F [4] and plant light-harvesting complex [5,6]. Electron micrographs of such images have provided the vital
phase information for 3D-structure determination of bacteriorhodopsin [7] and plant lightharvesting complex [8] at a resolution around 3.5 •~. These 2D crystals were supported by carbon film and preserved in a thin film of glucose or tannin, rather than in vitrified water. Thin 3D crystals imaged at a resolution of 6 ,~ and higher include those of CaZ+-ATPase [9], crotoxin [10] and of the viral helix-destabilizing gene product 32 (gp32) [11]. These were embedded in vitrified buffer but usually also supported by a continuous carbon film. Specimens that do not form extensive 2D or 3D crystals are usually prepared for electron cryo-microscopy in an unsupported film of vitrified acjueous buffer. However, image resolutions of 10 A or better have been difficult to achieve in this way. Jeng et al. [12] determined the structure of TMV at 10 A resolution, and Unwin [13] that
0304-3991/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-3991(94)00075-X
142
M Cyrklaff ~ Kiihlbrandt/UItramicroscopy 55 (1994) 141-153
of acetylcholine receptor to 9 A resolution by electron microscopy of frozen-hydrated tubular crystals. Even though it is inherently more difficult to observe high-resolution details in these specimens than with extensive 2D and 3D crystals, the large difference in resolution suggests that factors related to the specimen itself and to its interaction with the electron beam are responsible. Several of these factors, including specimen charging, beam-induced movement and radiation damage have been addressed in a recent article by Henderson [14]. In this study we investigate the effect of the structure of the medium surrounding the specimen, using images of tobacco mosaic virus (TMV) and catalase both in vitrified suspension and in films of cubic ice, formed by controlled devitrification. High-resolution electron diffraction patterns of T M V enable us to determine the contribution of each Fourier component to a perfect image of TMV. Our results indicate that high-resolution contrast is stronger for devitrified specimens than for otherwise identical vitrified specimens and that, therefore, cubic ice is preferable to vitrified water at least for the test specimens investigated by us. Images of the ice lattice recorded in a side-entry cryo-transfer holder indicate that cubic-ice-embedded specimens are stable to 1 A.
grids prepared in this way were used for specimen preparation without further conditioning by glow discharging. The properties of the carbon surface remained stable over a period of several weeks. Specimens of vitrified water and cubic ice were prepared from 10raM aqueous solution of NaCI or 10mM Tris buffer (pH 7.2), since pure water does not form thin films easily. Thin, 3D crystals of bovine liver catalase (Boehringer, Mannheim, Germany), recrystallized according to Wrigley [16] and tobacco mosaic virus (TMV) (provided by Dr. J. Witz, Strasbourg) were used for resolution tests in vitrified water and cubic ice. T M V was at a protein concentration of about 15 m g / m l in sodium phosphate buffer. Cryo-hydrated specimens were prepared as described by Dubochet et al. [17] with minor modifications. An aliquot (2-3 /xl) of the specimen suspension was applied to the carbon side of the grid mounted in the forceps of a guillotine for rapid freezing. Excess liquid was blotted off with filter p a p e r (Whatmann no. 1), and the grid was immediately plunged into liquid ethane cooled with liquid nitrogen to a temperature of about 170°C. The grid was mounted under liquid nitrogen in a side entry specimen holder [18] (Oxford Instruments, CT-3500 Cyro-Transfer System), and transferred into a JEOL-2000EX electron microscope.
2. Materials and methods
2_2. Controlled deuitrification 2.1. Preparation of samples for EM observation Thin, perforated films of cellulose-acetatebutyrate (Aldrich-Steinham, Germany) with an average hole size of 2 to 4/.~m were prepared as described by Fukami and Adachi [15], deposited on 200 mesh copper grids and coated with a 200 layer of carbon. Grids were placed overnight onto a stack of filter paper soaked with acetone in order to dissolve the cellulose acetate film. This treatment made the carbon slightly hydrophobic, as suggested by the minimal contact surface of water with the edge of the carbon film. This helped to obtain a thin and uniform film of aqueous buffer in the holes of the carbon. Micro-
Vitreous water was converted into ice by warming the specimen in the electron microscope up to - 115°C for 15 rain by passing an electrical current through the heating coils of the specimen holder. The heating current was then turned off to allow the specimen to cool to -168°C. The total time the specimen was at a temperature above the devitrification point of - 1 3 0 ° C was about 20 rain. This converted most of the vitrified water to cubic ice, but some remained in the vitreous state. For complete devitrification, the specimen was heated to a temperature above that of hexagonal ice formation (-90°C)_ At this temperature, ice
M. Cyrklaff, V~ Kiihlbrandt / Ultrarmcroscopy 55 (1994) 141-153
sublimates in the high vacuum of the electron microscope. The specimen was therefore taken out of the microscope, placed in the cryo-transfer unit, and the grid was warmed up to a temperature of - 7 5 ° C for about 10 rain. Then the specimen was cooled again to -168°C, and placed back into the microscope. Contamination from water vapor condensing on the specimen surface was minimized by liquid-nitrogen-cooled shutter blades on both sides of the specimen holder, and by passing a stream of cold, dry nitrogen over the tip of the holder. We did not observe any ice contamination after this process, and apparently no water was lost due to sublimation.
2.3. Electron microscopy Specimens were observed and images were recorded at 200 kV accelerating voltage. The instrument was equipped with a LaB 6 cathode, with a high-resolution polepiece (SHP-20, C s = 1.9 mm), and with a twin blade anticontaminator, similar to that of Homo [19], cooled externally with liquid nitrogen to a temperature of - 175°C. Images were recorded either without an objective lens aperture, or with a 100o~m aperture having a resolution cut-off below 2 A. Images of TMV and catalase were recorded at a magnification of 60000 X, with minimal electron irradiation of 10( _+2) e ,~-2, plus roughly 0.5 e ,~-2 for searching at low magnification. The objective lens was focused on the phase grain of the carbon support film close to the area of interest. The objective lens current was adjusted to give an underfocus of approximately 1500 ,&. The objective lens astigmatism was corrected before recording each image. Micrographs were recorded when the specimen drift had subsided to less than 0.3 ,& s - 1 as judged by observation on an image intensifier (Gatan, model 622 Fiber Optically Coupled TV System), with an exposure time of either 0.5 or 1 s. After recording each image, the actual defocus was estimated by eye on a piece of carbon film adjacent to the imaged area as inaccuracies frequently arise from the lack of planarity of holey carbon film. Only images with an actual underfocus close to 1500 ,& were used for further processing.
143
Rafts of aligned TMV rods were prepared from a concentrated suspension of TMV (15 mg/ml). A drop of suspension was placed on the grid, washed with distilled water and kept for a few minutes at room temperature in air to further concentrate the specimen by evaporation. After blotting the excess suspension, the concentrated TMV rods aligned within the holes of the support film, forming ordered rafts measuring up to 6 p,m across. Electron diffraction patterns of TMV were collected with a camera length of 1500 mm, and o 2 with an electron dose of up to 1 e A , using the low-dose procedure described by Kiihlbrandt [20]. Electron diffraction patterns of catalase crystals were collected with the same conditions as of TMV rafts. For selected crystals, two diffraction patterns were recorded, before and after devitrification of the surrounding buffer. Low-dose conditions were also used for imaging ice crystals, at magnifications ranging from 40000 to 250000 x . With exposure times of 1 and 0.5 s, the total dose for individual images varied between 5 e ,~-2 (40000 x ) to 100 e ,~-2 (250000 x ). Electron diffraction patterns of ice were collected with a camera length of 1500 mm, at an electron dose of less then 1 e ,~-2. Images and electron diffraction patterns were recorded on K O D A K SO-163 EM film and developed in full strength K O D A K D-19 developer for 12 min at room temperature. The optical density of images ranged from 0.3 to 0 . 6 0 D .
2.4. Image analysis Images were first evaluated by optical diffraction. For images of TMV, a slot aperture was adjusted to include either one or three parallel TMV rods. Images of the ice lattice were selected for high-resolution optical diffraction in all directions. The dimensions and distribution of ice crystals were analyzed by scanning the negative in the beam diffractometer with a small aperture, corresponding to (100 ,~)2 at the specimen. Selected micrographs were digitized on a Perkin-Elmer 1010M flat-bed microdensitometer, with a pixel size of 10 p,m (corresponding to 1.67 ,~ at the specimen). Areas for scanning were selected by optical diffraction, and marked on the
144
M. Cyrklaff W Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
negative. Two of the best diffraction patterns of T M V rafts were also digitized with a pixel size of 50 /zm. They were further processed with programs kindly provided by P. Metcalf (EMBL, Heidelberg). Both the integral intensity and the peak intensity of each layer line were determined. For the I n t 6 / I n t 0 ratio, the integral intensity in optical density units was used to derive the number of electrons contributing to the signal of the 6th layer line at 11.5 ,~ resolution, after subtracting the background, and compared to the estimated total number of electrons per illuminated area of a T M V raft, which contributed to the electron diffraction pattern. Fourier transforms of T M V and catalase images were generated using image processing programs kindly supplied by R. Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK). T M V image intensities were calculated by squaring peak amplitudes on each layer line, and subtracting the averaged and squared local background. For the image transform, the ratio of I n t 6 / I n t 0 was taken as the ratio of highest intensity of the 6th layer line and the calculated intensity at the origin.
3. Results
3.1. Images of thin films of ice Thin films of vitrified water or buffer with an estimated thickness of 200 to 1000 A on holey carbon film were produced by a routine method [21] and subjected to controlled devitrification by which the vitrified water was converted to cubic ice. The size of the ice crystallites varied somewhat with experimental conditions. It was usually in the range of 200 to 1000 A, but larger crystallites of up to 1 ~ m in diameter were also observed mostly in specimens devitrified at a temperature of -75°C. Individual crystallites were randomly oriented. In defocused, low-magnification electron micrographs, ice crystals which happened to be in a reflecting position diffracted electrons strongly and were imaged with high contrast, giving the ice film a coarse, grainy appearance (Fig. la). Images of films devitrified at o
atomospheric pressure (see Section 2) and recorded at high magnification (Fig. lb) revealed that these were entirely crystalline. Crystallites were in contact at mutual grain boundaries rather than being separated by regions of uncrystallized, vitrified buffer. Images of individual crystallites recorded close to focus at 250000 × magnification and an incident dose of 100 e A 2 were examined by optical diffraction. Most images showed several different lattice types. The (111) reflection of the cubic-ice lattice at 3.66 A was the most prominent. Frequently, the second order of this reflection at 1.83 A resolution was visible. About 10% of the optical diffractograms of cubic-ice lattices also showed the third order at 1.25 A and, occasionally, the fourth order at 0.915 A (Fig_ lb). Other commonly observed reflections were at 1.72, 1.91, 2.24 and 3.89 A. The first and the last index on the hexagonal lattice, the other two belong to the cubic lattice [17]. Nearly all crystals obtained by devitrification above - 130°C showed hiogh-resolution reflections between 2.24 and 1.72 A in the optical diffractogram. Reflections of similar resolution were observed in specimens devitritied at - 7 5 ° C and atmospheric pressure. Specimens could also be devitrified by irradiation with high electron doses. However, this gave reflections between 3.66 ando 2.24 A only rarely, and very rarely above 2.24 A, presumably due to radiation damage.
3.2. Ice-embedded catalase Specimens of thin 3D crystals of catalase were prepared for electron microscopy in a thin film of vitrified buffer. This was then devitrified as before so that the catalase crystals were embedded in a thin film of cubic ice. A typical specimen area is shown in Fig. 2b. In areas where the film is thicker, individual ice crystallites overlap with the catalase crystals (Fig. 2a). Electron diffraction patterns of catalase crystals on the same grid before and after devitrification extended to 2 A resolution (results not shown), demonstrating that the formation of ice crystals during devitrification did not disrupt high-resolution detail. Low-dose images of thin
NL Cyrklaff, F¢~Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
145
Fig. 1. Film of unsupported cubic ice_ (a) Low-magnification image showing the characteristic granular appearance. (b) High-magnification image (electron-optical magnification: 250k x ) of a single crystallite measuring about 1000 A, across, showing lattice fringes (111) of cubic ice (1¢ = 3.66 ,~). The inset shows the corresponding optical diffraction pattern with I c (444) = 0.92 A (arrow). Cubic ice was produced by controlled devitrification of water in the electron microscope, and imaged with a dose of approximately 100 e ,~-2. Space bar (a) 2 ~zm and (b) 100 A,.
146
M. Cvrklc(~ W. KtThlbrandt / UItramicroscopy 55 (1994) 141 153
12,11 )
12~8
Fig. 2. (a) Thin 3D catalase crystal embedded in ice. (b) High-magnification image showing the catalase lattice, recorded with 10 e 2 at magnification 60k × . (c) Optical diffraction pattern of a catalase crystal similar to the one shown in (a). (d) Part of the calculated, background-corrected Fourier transform of the same image, showing spots on the reciprocal lattice beyond the 8th order, extending to 5.4 A. The cell dimensions of beef liver catalase crystals are a 69 A, b = 173.5 ,~ [31]. Space bars (a) I # m and (b) 500 ,~
M. Cyrklaff, ~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
catalase crystals were examined by optical diffraction and showed reflections to 5.4 A (Fig. 2c and 2d). Images of ice-embedded specimens gave somewhat sharper reflections and the success rate of recording high-resolution images was higher than with vitrified specimens.
147
OD 1.5-~
i
i
I
i
i
I
i
0,02
0
1.0
3.3. Tobacco mosaic c'irus As a second test specimen for high-resolution imaging we used tobacco mosaic virus in vitrified buffer and cubic ice.
0.5
3.3.1. Electron diffraction of TMV In concentrated suspension, T M V particles tend to align, making rafts measuring up to 6 / x m in diameter. Occasionally such rafts are suffi-
0 0 04
I
0.02
I
I
0.04
-1
~,
Fig. 4. Intensity profile of the meridional range of the 6th layer line in the electron diffraction pattern of TMV shown in Fig. 3. The modulation of the intensity shows that the TMV particles were well-aligned.
ciently well ordered for high-resolution electron diffraction [22]. The layer line intensities recorded in the diffraction pattern thus represent an average over many hundreds of particles. An example of an electron diffraction pattern of T M V in vitrified buffer is shown in Fig. 3. The 31st layer line is visible at the meridian, corresponding to a resolution of 2.3 A in this direction. In the equatorial direction, reflections are more diffuse and extend to roughly 5 ,~. The intensity profile of the 6th layer line, at 11.5 ,& resolution at the meridian (Fig. 4), indicates that the particles were well oriented within the raft. Scaled values of maximal, background-corrected electron diffraction intensities on each layer line are listed in Table 1. The ratio of maximal intensities on the 3rd layer line at 23.0 ,~ resolution and the 6th layer line was 0.56.
Fig. 3. Electron diffraction pattern of a TMV raft_ Camera length 1500 mm, dose below 1 e ~ - 2 . The lower half shows the low-resolution 3rd and 6th layer lines, as used for calculating the intensity ratio of them. Rings are due to subtraction of rotationally averaged background_
3.3.2_ Images of TMV Images of T M V were recorded in vitrified and devitrified buffer. In order to optimize contrast in the 7 - 1 2 A resolution range, images were taken at an underfocus of approximately 1500 to 2000 •~. At this defocus, with an accelerating voltage of 200 kV and C~ = 1.9 mm, the first C T F minimum occurs at a spatial frequency of (6.0 ~,) 1 to (7.2 ~,)-t. Since low-resolution contrast is weak under
h/L Cyrklaffl W Kiihlbrandt / Ultramicroscopy 55 (1994) 141 153
148
these conditions, the virus particles were virtually invisible by eye on the electron micrographs. The rods could, however, be located precisely by optical diffraction, scanning the negatives with a rectangular aperture with a width corresponding to that of the T M V particles. Of the images which were properly focused, a total of about 150 of devitrified and about 100 of vitrified T M V specimens were evaluated by optical diffraction. The 6th layer line of the T M V diffraction pattern, corresponding to a resolution of 11.5 A, was detectable in about 55% of devitrifled, and in about 40% of vitrified samples. The 3rd layer line was comparatively weak, due to the contrast transfer function (CTF) at this defocus. T M V particles showing detail beyond the 8th layer line were found only on devitrified specimens. The best ones of these showed the 9th layer line, at a resolution of 7.7 ,&. In one instance, the 12th layer line, at approximately 5.75 ,~, was visible by optical diffraction (Fig. 5), using an aperture corresponding to the width of three parallel T M V rods. With this larger aperture, the observed layer line intensities were stronger by a factor of roughly 5 compared to a single T M V particle. In the Fourier transform of this area, the 10th, l l t h and 12th layer lines had peak intensities above background of, respectively, 8, 9 and 14 after scaling to the 6th layer line as in Table 2.
Six separate areas on the four best images (three of devitrified, one of a vitrified specimen) were digitized on a microdensitometer after masking the area on the negative, with the long axis of the particles parallel to the X-scan direction. The Fourier transforms of each digitized area were then examined for maximum intensities along each layer line. Results are shown in Table 2_
3.3.3. Assessment of TMV image quality
o
The electron diffraction intensities of T M V provide an objective criterion for assessing the quality of images. In an ideal image, the intensities in the Fourier transform should be identical to those measured by electron diffraction. In reality, however, the intensities in particular of highorder terms are severely reduced by specimen-related effects, such as inelastic scattering, radiation damage, sample movements and charging. In addition, the molecular transform is modulated by the contrast transfer function (CTF) of the electron microscope. If the CTF is known, then corrected structure factor intensities can be calculated by multiplying each image intensity with the inverse of the CTF for a particular spatial frequency. In practice, the CTF is determined from the positions of the Thon-rings in the optical diffraction pattern, i.e_ the spatial frequencies
Table 1 Structure factor intensities in the electron diffraction pattern of cryo-hydrated TMV rafts Layer line
1
2
3
4
5
6
7
8
9
10
11
12
Intensity
n.d.
n.d.
56.3
20.1
26.7
100.0
42.1
17.9
17.8
14.5
13.2
18.8
Layer line
13
14
15
16
17
18
19
20
21
22
23
24
Intensity
11.4
11.6
35.0
13.8
11.4
21.5
11.2
12.3
13.0
14.6
11.9
13.3
Layer line
25
26
27
28
29
31)
31
Intensity
11.0
11.4
11.3
11.0
10.8
11.1
9.6
Background-corrected structure factor intensities of the TMV electron diffraction pattern. The maximum intensity for each layer line is shown, scaled to the 6th layer line. The first and second layer lines were not visible due to the high background close to the origin.
M. Cyrklaff, I~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
This is less and less likely at increasing magnification. However, the best image of T M V in vitrified buffer (no. 3296) shows an area of carbon film which allowed us to determine the first minimum of the CTF and thus to correct the observed intensities. The CTF was calculated assuming 7% amplitude contrast as found for unstained weak-phase, weak-amplitude specimens imaged in the microscope with 100 kV electrons [23]. At an accelerating voltage of 200 kV, the amplitude contrast would be somewhat lower, and this would slightly increase the correction factor. The intensities were not corrected for the envelope function which is difficult to estimate. The envelope would decrease the high-resolution intensities and therefore lower the correction factor. The intensity ratios of the 3rd versus 6th layer lines for three different particles in this image ranged from 2.9 to 4.9 (Table 2). This is much worse than the ideal value of 0.56 obtained by electron diffraction, indicating a major loss of contrast at a resolution of 11.5 A and above. However, these values compare favorably with others reported previously for T M V in vitrified suspension [12,14,24]. Only one of the 3 best images of T M V in cubic ice contained any carbon film, so that the CTFcorrected structure factor intensities could not be determined exactly for all of them. Since we aimed to record all images at defocus between 1500 and 2000 A, we can assume, however, that the C T F is similar for all images and close to maximal for the 6th layer line at 11.5 ,~ resolution. Making this assumption, we obtained intensity ratios of the 3rd versus the 6th layer line of 2.3 (_+0.56) for a single T M V particle. For the Fourier transform of three parallel T M V rods in the same image area, this ratio improved to 1.8 (_+0.3) as a result of the increased signal/noise ratio. Cubic ice therefore seems to be preferable to vitrified water as a medium for high-resolution electron cryo-microscopy. Table 2 also shows the ratio of the strongest diffracted intensity (on the 6th layer line) to the intensity at the origin, as a measure of absolute contrast. For the images, these ratios range from 2 x 10 -.7 to 3.5 x 10 -~, which is roughly three orders of magnitude lower than the ratio estio
Fig. 5. Optical diffraction pattern of three parallel T M V rods, 0.2 Izrn long. The arrow points to the 12th layer line, corresponding to a resolution of 5 75 ,~. The layer lines are appearing on one side of the meridian only. This is characteristic for one-sided helices and may indicate partial distortion of the particles for example by protrusion through the water surface and partial dehydration.
for which the contrast is zero. The contrast of biological specimens in vitreous water or cubic ice is too low to give rise to detectable Thon rings. However, in favorable circumstances, a good estimate of the C T F can be obtained if the imaged area includes some carbon film.
149
n.d.
Electron diJ]kaction
n.d.
43.2 10.0 47.2
12.3 25.3 55.2
0.063
2
56.3
90.5 48.0 45.6
78.8 61.8 103.0
0.185
3
2/).1
27.2 9.4 25.6
9.0 19.3 24.5
0.397
4
26.7
57.5 6.1 49.4
11.2 17.2 28.2
0.722
5
100.0
100.0 100.0 100.0
100.0 100.0 100.0
0.884
6
42.1
40.2 7.0 20.3
6.7 19.8 23.5
0.640
7
17.9
29.6 3.9 19.6
10.0 13.8 28.8
0.137
8
17.8
19.4 5.0
-
0.032
9
0.56
0.91 0.48 0.46
0.79 0.62 1.03
Ratio 3rd/6th
4.35(+ / 2.29(+/ 2.19
3.77 2.96 4.92
Ratio 3rd/6th (CTF) 2 corrected
0.56) * 0.30)*
5.2x 10 4
4.3 x 10 s 2.2x10 7 3.5 x l0 s
2.0x10 7 9.0x 10 ~ 6.6x 10 -8
Origin/6th layer line intensity
Background-corrected intensities were scaled to the 6th layer line intensity before CTF correction. The first minimum of the CTF was obtained by optical diffraction of carbon film in image #3296, and calculated for 200 kV accelerating voltage, with the objective lens spherical aberration value equal to 1.9 mm, and assuming 7% amplitude contrast [23], without including an envelope decay function. This CTF was used to correct the intensities for this image, as well as for the ice-embedded images indicated by *. Estimated error margins for the correction are _+ 13%, due to the uncertainty of defocus. The CTF for image #2739 was measured in the same way and found to be very similar. Selected electron diffraction intensities from Table 1 are given in the bottom row for comparison. The ratio of intensity of the 6th layer line to the intensity at the origin was derived from image transforms of TMV. The ratio for electron diffraction was calculated from the number of electrons calculated from the total integrated intensity above background of the 6th layer line to the estimated total number of electrons used to record the diffraction pattern.
43.8 8.5 35.2
12.5 21.1 19.8
0.014
(CTF) 2
1
Layer lines
2908 2912 2739
Ice
3296 1 3296 2 3296 3
Vitrified
Image number
Table 2 Structure factor intensities in the computed Fourier transforms of TMV in cubic ice and vitrified water
',.a t~a
I
4k
©
M. Cyrklaff 144 Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
mated from an electron diffraction pattern (5.2 × 10-4). However, the ratio is 5 times higher for image transforms of three parallel T M V rods (1.2 × 10-6), due to the higher signal/noise ratio.
4. Discussion
The frozen hydrated preparation method should be ideal for preserving high-resolution detail of any biological specimen. However, it is striking that near-atomic image resolution has, so far, been achieved more reliably with specimens in glucose and tannin, rather than in vitrified buffer. This indicates that the problems associated with high-resolution imaging are at least in part due to the water support. One of these problems is caused by small movements of the specimen and the supporting film as they absorb energy from the electron beam. It may be expected that a film composed of small, tightly interlaced crystals is mechanically more rigid and hence less prone to move under the beam. The results presented here demonstrate that this is indeed the case. The fact that electron micrographs of the cubic-ice lattice diffracting to 1 - 2 / ~ in the laser b e a m can be recorded routinely indicates clearly that it is a rigid, mechanically stable support film. The crystal lattice makes it easy to check whether or not specimen movement has occurred while an image was recorded and to test the performance of the electron microscope and of the specimen holder at conditions used for cryo-microscopy. It is possible that small specimen movements and the build-up of charges occur mainly in the initial period of exposure and that the specimen stabilizes after about 10 e ~ - 2 . This may account for the high resolution of the ice-lattice images which were recorded with a dose of 100 e ,&-2 which is more than 10 times higher than what can be used in biological electron cryo-microscopy at liquid nitrogen temperature. Nevertheless, the success rate of recording 11.5 A images of T M V particles indicates the increased mechanical stability of cubic ice compared to vitrified buffer. Ice-crystal formation can cause severe damage
151
to biological samples, primarily because the growing ice crystals exclude solute molecules and ions, thus causing osmotic stress in the remaining volume. The disruption of the hydration layer around macromolecules could be another cause of damage. Because freezing is a strongly exothermic process, it takes place generally at a temperature only slightly below the freezing point of 0°C. Freezing damage is avoided when water is vitrified, namely when it is cooled so rapidly that it has no time to start forming ice crystals before the viscosity becomes so high that any movements are practically blocked [25]. In the present work, ice-crystal formation takes place under conditions which are very different from the usual freezing around 0°C in that water molecules immobilized in the vitrified state are rewarmcd to a temperature where crystallization occurs. Apparently, crystallization of water under these conditions involves only minimal molecular displacements, and we have shown that biological macromolecules are unaffected by this process. It has been observed before that some crystalline biological specimens are well preserved even in presence of large ice crystals [26]. This was thought to be due to the fact that the crystals form from amorphous water [27], consistent with the results presented here_ Taylor and Glaeser [28] found that thin crystals of catalase diffract to 2.8 A even after freezing in liquid nitrogen which results in the formation of large ice crystals. Catalase crystals have a high protein packing density and presumably contain little liquid water which may explain why they are less sensitive to freezing damage. However, it is well known that freezing in liquid nitrogen damages single particles and other specimens with a high water content [17]. The procedure applied by us requires careful control of the specimen temperature_ If this rises above -90°C, where water begins to sublimate rapidly, there is a risk of freeze-drying the specimen. Our images of T M V and catalase show that this has not occurred since they are clearly embedded in a continuous film of microcrystalline buffer (Fig. 2a). The T M V images were recorded with an objective lens defocus of 1500 to 2000 A and thus much closer to focus than in other studies
152
M. Cyrklaff V~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141 153
[12,14,24]. At this defocus, the CTF maximum is close to the 6th layer line (in image 3296 located below the first maximum with a value of 0.884), whereas the 9th and higher layer lines lie beyond the first minimum. Low-resolution frequencies are de-emphasized so that the typical virus features (a rod of 180 A width, with 23 A striations) are not visible by eye of the micrographs. However, single helical rods of T M V can be easily localized by optical diffraction. In this way, structural information in the l 1.5 to 7.5 ,~ resolution range can be recorded with single T M V particles. Clearly, averaging over several T M V images of this quality w o u l d greatly i m p r o v e the signal/noise ratio. Following a suggestion by Henderson [14], we used the ratio of image intensities of the 3rd versus the 6th layer line to assess the quality of an image. This ratio compares information about molecular envelope (3rd layer line) with that about the arrangements of secondary structure within the protein (the 6th layer line and higher). This ratio was calculated both for images and for the electron diffraction patterns_ We find that for the electron diffraction patterns the ratio of the 3rd versus the 6th layer line is higher by a factor of 1.5 compared to X-ray diffraction, exactly as predicted by Henderson [14]. This appears to be due to the increased low-resolution contrast of T M V in ice (which has a density of 0.92 g ml J) compared to T M V in water. In the computed transforms of T M V images, corrected for the contrast transfer function, the ratios of the 3rd to 6th layer lines intensities are on average six times higher than the reference, whereas values reported by Henderson [14] for the T M V images collected in various electron microscopes at considerably higher defocus are between 11 and 35. This is partially due to the fact that our images were closer to focus. We realize, however, that images in which the particles are not visible by eye will be useful only if their position can be detected in some other way, e.g. by optical diffraction, or by recording a second image at higher defocus. We hope that these studies will contribute towards imaging fully hydrated, crystalline and non-crystalline biological specimens at high reso-
lution. At present, the absolute contrast of our best image of a single particle is about 1000 times worse than in a perfect image, which suggests that very large improvements can be expected, once the remaining problems have been identified and overcome. This should be possible in the near future, both by improvements in instrumentation and in specimen preparation. Instrumental improvements include higher acceleration voltage, lower specimen temperature, the use of energy filters [29], and objective lens correctors [30]. Improvements in specimen preparation may include evaporating a conducting layer onto one or both ice surfaces to reduce specimen charging and movement.
5. Conclusions (1) We have shown that thin films of cubic ice, produced by controlled devitrification of vitreous water, provide a rigid and stable support suitable for high-resolution imaging of biological macromolecules. The cubic-ice film is stable to within 1 A. (2) The ice lattice can be used as a convenient test object to assess the performance of the electron microscope and the specimen holder used for cryo-imaging. (3) Biological macromolecules embedded in cubic ice were imaged at higher resolution and with better signal/noise ratio than comparablc specimens embedded in vitreous water. (4) Thin crystals of catalase embedded in vitrified medium or in cubic-ice-diffracted electrons to 2 A, indicating that the specimen was not damaged by the formation of ice crystals during devitrification. (5) Electron diffraction patterns of oriented T M V rafts provided a reference for assessing the quality of single-particle images. Image intensities between 23 and 11.5 A resolution decay roughly 6 times more rapidly than electron diffraction intensities, indicating that large improvements in image quality are still required. o
M. Cyrklaff, l~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
Acknowledgements We gratefully acknowledge Richard Henderson a n d J a q u e s D u b o c h e t for critical c o m m e n t s , and Kevin Leonard for helpful suggestions. M.C. specially thanks Bjcrn Johansen and Norbert Roos for support and valuable discussions during his visits to O s l o U n i v e r s i t y .
References [1] R. Henderson, J.M. Baldwin, K.H. Downing, J. Lepault and F. Zemlin, Uhramicroscopy 19 (1986) 147. [2] J.M. Baldwin, R. Henderson, E. Beckmann and F Zemlin, J. Mol. Biol. 202 (1988) 585 [3] B.K. Jap, P.J. Walian and K. Gehring, Nature 350 (1991) 167. [4] H.J. Sass, G. Bfildt, E. Beckmann, F. Zemlin, M. van Hill, E. Zeitler, J.P. Rosenbusch, D.L. Dorset and A. Massalski, J. Mol. Biol. 209 (1989) 171 [5] W. Kfihlbrandt and K.H_ Downing, J. Mol. Biol. 207 (1989) 823. [6] DN. Wang and W. Kiihlbrandt, J Mol. Biol. 217 (1991) 691. [7] R. Henderson, J.M_ Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann and K.H. Downing, J. Mol. Biol. 213 (1990) 899. [8] W. Kiihlbrandt, D.N. Wang and Y. Fujiyoshi, Nature 367 (1994) 614. [9] D.L. Stokes and N.M. Green, J. Mol. Biol. 213 (1990) 529. [10] J. Brink, W. Chiu and M. Dougherty, Ultramicroscopy 46 (1992) 229.
153
[11] R. Grant, M.F. Schmidt and W. Chiu, J. Mol. Biol. 217 (1991) 551. [12] T.-W. Jeng, R.A. Crowther, G. Stubbs and W. Chiu, J_ Mol. Biol. 205 (1989) 251. [13] N. Unwin, J. Mol. Biol. 229 (1993) 1101. [14] R. Henderson, Ultramicroscopy 46 (1992) 1. [15] A. Fukami and K. Adachi, J. Electron Microsc. 14 (1965) 112. [16] N.G. Wrigley. J. Ultrastruct. Res. 24 (1968) 454. [17] J. Dubochet, M. Adrian, J.-J. Chang, J.-C. Homo, J. Lepault, A.W. McDowall and P. Schultz, Q. Rev. Biophys. 21 (1988) 129. [18] R. Henderson, C. Raeburn and G. Vigers, Ultramicroscopy 35 (1991) 45. [19] J.-C. Homo, J. Microscopy 136 (1984) 337. [20] W. Ki~hlbrandt, J. Mol. Biol_ 202 (1988) 849. [21] M. Adrian, J. Dubochet and A.W. McDowall, Nature 308 (1984) 32. [22] T.-W. Jeng and W. Chiu, Ultramicroscopy 23 (1987) 61. [23] C. Toyoshima and N. Unwin, Ultramicroscopy 25 (1988) 279. [24] R.R. Schr6der, W. Hofmann and J.-F. Menetret, J. Struct. Biol. 105 (1990) 28. [25] F. Franks, Properties of aqueous solutions at subzero temperatures, in: Water: A Comprehensive Treatise. Water and Aqueous Solutions at Subzero Temperatures, Ed. F. Franks (Plenum, New York, 1982) p. 215. [26] R.A. Milligan, A. Brisson and P.N.T. Unwin, Ultramicroscopy 13 (1984) 1. [27] J. Dubochet, K. Richter, H.-V. Roy and A.W. McDowall, Scanning Microsc. Suppl. 5 (1991) 11. [28] K.A. Taylor and R.M. Glaeser, J. Ultrastruct. Res. 55 (1976) 448. [29] R.R. Schr6der, J. Microscopy 166 (1992) 389. [30] H. Rose, Optik 85 (1990) 19. [31] P.N.T. Unwin, J. Mol. Biol. (1975) 235.
Ultramicroscopy 55 (1994) 141-153
High-resolution electron microscopy of biological specimens in cubic ice Marek Cyrklaff, Werner Kiihlbrandt European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Received 1 December 1993; in final form 4 May 1994
Abstract
Images of two biological test specimens, catalase and TMV, were recorded in cubic ice, prepared by controlled devitrification at -130°C and -75°C or in vitrified buffer. Cubic ice provides a rigid support for biological specimens which is stable under the electron beam to within about 1 ,~, as shown by images of the ice lattice. Neither catalase nor TMV were disrupted by the crystallization of vitrified water. Electron diffraction patterns of highly oriented rafts of TMV extending to 2.3 ,& resolution were used to judge the quality of TMV images. Structural features at 5.75 ,& (TMV) and 5.4 ,& (catalase) were observed directly by optical diffraction. The reproduction of high-resolution details of TMV was better in cubic ice, and the success rate for recording good images was higher than in vitrified medium.
1. Introduction
The structure of biological macromolecules and macromolecular assemblies can be determined by electron microscopy at atomic or near-atomic resolution, provided that it is possible to record images in which high-resolution features of the object are faithfully reproduced. This requires (a) a n instrument which is capable of recording atomic resolution images of biological specimens and (b) that the object is preserved in its native, hydrated form. So far, images with resolutions between 3 and 4 A have been recorded primarily of two-dimensional (2D) and thin three-dimensional (3D) protein crystals. 2D crystals imaged at high resolution include bacteriorhodopsin [1,2], the bacterial porins PhoE [3], O m p F [4] and plant light-harvesting complex [5,6]. Electron micrographs of such images have provided the vital
phase information for 3D-structure determination of bacteriorhodopsin [7] and plant lightharvesting complex [8] at a resolution around 3.5 •~. These 2D crystals were supported by carbon film and preserved in a thin film of glucose or tannin, rather than in vitrified water. Thin 3D crystals imaged at a resolution of 6 ,~ and higher include those of CaZ+-ATPase [9], crotoxin [10] and of the viral helix-destabilizing gene product 32 (gp32) [11]. These were embedded in vitrified buffer but usually also supported by a continuous carbon film. Specimens that do not form extensive 2D or 3D crystals are usually prepared for electron cryo-microscopy in an unsupported film of vitrified acjueous buffer. However, image resolutions of 10 A or better have been difficult to achieve in this way. Jeng et al. [12] determined the structure of TMV at 10 A resolution, and Unwin [13] that
0304-3991/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-3991(94)00075-X
142
M Cyrklaff ~ Kiihlbrandt/UItramicroscopy 55 (1994) 141-153
of acetylcholine receptor to 9 A resolution by electron microscopy of frozen-hydrated tubular crystals. Even though it is inherently more difficult to observe high-resolution details in these specimens than with extensive 2D and 3D crystals, the large difference in resolution suggests that factors related to the specimen itself and to its interaction with the electron beam are responsible. Several of these factors, including specimen charging, beam-induced movement and radiation damage have been addressed in a recent article by Henderson [14]. In this study we investigate the effect of the structure of the medium surrounding the specimen, using images of tobacco mosaic virus (TMV) and catalase both in vitrified suspension and in films of cubic ice, formed by controlled devitrification. High-resolution electron diffraction patterns of T M V enable us to determine the contribution of each Fourier component to a perfect image of TMV. Our results indicate that high-resolution contrast is stronger for devitrified specimens than for otherwise identical vitrified specimens and that, therefore, cubic ice is preferable to vitrified water at least for the test specimens investigated by us. Images of the ice lattice recorded in a side-entry cryo-transfer holder indicate that cubic-ice-embedded specimens are stable to 1 A.
grids prepared in this way were used for specimen preparation without further conditioning by glow discharging. The properties of the carbon surface remained stable over a period of several weeks. Specimens of vitrified water and cubic ice were prepared from 10raM aqueous solution of NaCI or 10mM Tris buffer (pH 7.2), since pure water does not form thin films easily. Thin, 3D crystals of bovine liver catalase (Boehringer, Mannheim, Germany), recrystallized according to Wrigley [16] and tobacco mosaic virus (TMV) (provided by Dr. J. Witz, Strasbourg) were used for resolution tests in vitrified water and cubic ice. T M V was at a protein concentration of about 15 m g / m l in sodium phosphate buffer. Cryo-hydrated specimens were prepared as described by Dubochet et al. [17] with minor modifications. An aliquot (2-3 /xl) of the specimen suspension was applied to the carbon side of the grid mounted in the forceps of a guillotine for rapid freezing. Excess liquid was blotted off with filter p a p e r (Whatmann no. 1), and the grid was immediately plunged into liquid ethane cooled with liquid nitrogen to a temperature of about 170°C. The grid was mounted under liquid nitrogen in a side entry specimen holder [18] (Oxford Instruments, CT-3500 Cyro-Transfer System), and transferred into a JEOL-2000EX electron microscope.
2. Materials and methods
2_2. Controlled deuitrification 2.1. Preparation of samples for EM observation Thin, perforated films of cellulose-acetatebutyrate (Aldrich-Steinham, Germany) with an average hole size of 2 to 4/.~m were prepared as described by Fukami and Adachi [15], deposited on 200 mesh copper grids and coated with a 200 layer of carbon. Grids were placed overnight onto a stack of filter paper soaked with acetone in order to dissolve the cellulose acetate film. This treatment made the carbon slightly hydrophobic, as suggested by the minimal contact surface of water with the edge of the carbon film. This helped to obtain a thin and uniform film of aqueous buffer in the holes of the carbon. Micro-
Vitreous water was converted into ice by warming the specimen in the electron microscope up to - 115°C for 15 rain by passing an electrical current through the heating coils of the specimen holder. The heating current was then turned off to allow the specimen to cool to -168°C. The total time the specimen was at a temperature above the devitrification point of - 1 3 0 ° C was about 20 rain. This converted most of the vitrified water to cubic ice, but some remained in the vitreous state. For complete devitrification, the specimen was heated to a temperature above that of hexagonal ice formation (-90°C)_ At this temperature, ice
M. Cyrklaff, V~ Kiihlbrandt / Ultrarmcroscopy 55 (1994) 141-153
sublimates in the high vacuum of the electron microscope. The specimen was therefore taken out of the microscope, placed in the cryo-transfer unit, and the grid was warmed up to a temperature of - 7 5 ° C for about 10 rain. Then the specimen was cooled again to -168°C, and placed back into the microscope. Contamination from water vapor condensing on the specimen surface was minimized by liquid-nitrogen-cooled shutter blades on both sides of the specimen holder, and by passing a stream of cold, dry nitrogen over the tip of the holder. We did not observe any ice contamination after this process, and apparently no water was lost due to sublimation.
2.3. Electron microscopy Specimens were observed and images were recorded at 200 kV accelerating voltage. The instrument was equipped with a LaB 6 cathode, with a high-resolution polepiece (SHP-20, C s = 1.9 mm), and with a twin blade anticontaminator, similar to that of Homo [19], cooled externally with liquid nitrogen to a temperature of - 175°C. Images were recorded either without an objective lens aperture, or with a 100o~m aperture having a resolution cut-off below 2 A. Images of TMV and catalase were recorded at a magnification of 60000 X, with minimal electron irradiation of 10( _+2) e ,~-2, plus roughly 0.5 e ,~-2 for searching at low magnification. The objective lens was focused on the phase grain of the carbon support film close to the area of interest. The objective lens current was adjusted to give an underfocus of approximately 1500 ,&. The objective lens astigmatism was corrected before recording each image. Micrographs were recorded when the specimen drift had subsided to less than 0.3 ,& s - 1 as judged by observation on an image intensifier (Gatan, model 622 Fiber Optically Coupled TV System), with an exposure time of either 0.5 or 1 s. After recording each image, the actual defocus was estimated by eye on a piece of carbon film adjacent to the imaged area as inaccuracies frequently arise from the lack of planarity of holey carbon film. Only images with an actual underfocus close to 1500 ,& were used for further processing.
143
Rafts of aligned TMV rods were prepared from a concentrated suspension of TMV (15 mg/ml). A drop of suspension was placed on the grid, washed with distilled water and kept for a few minutes at room temperature in air to further concentrate the specimen by evaporation. After blotting the excess suspension, the concentrated TMV rods aligned within the holes of the support film, forming ordered rafts measuring up to 6 p,m across. Electron diffraction patterns of TMV were collected with a camera length of 1500 mm, and o 2 with an electron dose of up to 1 e A , using the low-dose procedure described by Kiihlbrandt [20]. Electron diffraction patterns of catalase crystals were collected with the same conditions as of TMV rafts. For selected crystals, two diffraction patterns were recorded, before and after devitrification of the surrounding buffer. Low-dose conditions were also used for imaging ice crystals, at magnifications ranging from 40000 to 250000 x . With exposure times of 1 and 0.5 s, the total dose for individual images varied between 5 e ,~-2 (40000 x ) to 100 e ,~-2 (250000 x ). Electron diffraction patterns of ice were collected with a camera length of 1500 mm, at an electron dose of less then 1 e ,~-2. Images and electron diffraction patterns were recorded on K O D A K SO-163 EM film and developed in full strength K O D A K D-19 developer for 12 min at room temperature. The optical density of images ranged from 0.3 to 0 . 6 0 D .
2.4. Image analysis Images were first evaluated by optical diffraction. For images of TMV, a slot aperture was adjusted to include either one or three parallel TMV rods. Images of the ice lattice were selected for high-resolution optical diffraction in all directions. The dimensions and distribution of ice crystals were analyzed by scanning the negative in the beam diffractometer with a small aperture, corresponding to (100 ,~)2 at the specimen. Selected micrographs were digitized on a Perkin-Elmer 1010M flat-bed microdensitometer, with a pixel size of 10 p,m (corresponding to 1.67 ,~ at the specimen). Areas for scanning were selected by optical diffraction, and marked on the
144
M. Cyrklaff W Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
negative. Two of the best diffraction patterns of T M V rafts were also digitized with a pixel size of 50 /zm. They were further processed with programs kindly provided by P. Metcalf (EMBL, Heidelberg). Both the integral intensity and the peak intensity of each layer line were determined. For the I n t 6 / I n t 0 ratio, the integral intensity in optical density units was used to derive the number of electrons contributing to the signal of the 6th layer line at 11.5 ,~ resolution, after subtracting the background, and compared to the estimated total number of electrons per illuminated area of a T M V raft, which contributed to the electron diffraction pattern. Fourier transforms of T M V and catalase images were generated using image processing programs kindly supplied by R. Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK). T M V image intensities were calculated by squaring peak amplitudes on each layer line, and subtracting the averaged and squared local background. For the image transform, the ratio of I n t 6 / I n t 0 was taken as the ratio of highest intensity of the 6th layer line and the calculated intensity at the origin.
3. Results
3.1. Images of thin films of ice Thin films of vitrified water or buffer with an estimated thickness of 200 to 1000 A on holey carbon film were produced by a routine method [21] and subjected to controlled devitrification by which the vitrified water was converted to cubic ice. The size of the ice crystallites varied somewhat with experimental conditions. It was usually in the range of 200 to 1000 A, but larger crystallites of up to 1 ~ m in diameter were also observed mostly in specimens devitrified at a temperature of -75°C. Individual crystallites were randomly oriented. In defocused, low-magnification electron micrographs, ice crystals which happened to be in a reflecting position diffracted electrons strongly and were imaged with high contrast, giving the ice film a coarse, grainy appearance (Fig. la). Images of films devitrified at o
atomospheric pressure (see Section 2) and recorded at high magnification (Fig. lb) revealed that these were entirely crystalline. Crystallites were in contact at mutual grain boundaries rather than being separated by regions of uncrystallized, vitrified buffer. Images of individual crystallites recorded close to focus at 250000 × magnification and an incident dose of 100 e A 2 were examined by optical diffraction. Most images showed several different lattice types. The (111) reflection of the cubic-ice lattice at 3.66 A was the most prominent. Frequently, the second order of this reflection at 1.83 A resolution was visible. About 10% of the optical diffractograms of cubic-ice lattices also showed the third order at 1.25 A and, occasionally, the fourth order at 0.915 A (Fig_ lb). Other commonly observed reflections were at 1.72, 1.91, 2.24 and 3.89 A. The first and the last index on the hexagonal lattice, the other two belong to the cubic lattice [17]. Nearly all crystals obtained by devitrification above - 130°C showed hiogh-resolution reflections between 2.24 and 1.72 A in the optical diffractogram. Reflections of similar resolution were observed in specimens devitritied at - 7 5 ° C and atmospheric pressure. Specimens could also be devitrified by irradiation with high electron doses. However, this gave reflections between 3.66 ando 2.24 A only rarely, and very rarely above 2.24 A, presumably due to radiation damage.
3.2. Ice-embedded catalase Specimens of thin 3D crystals of catalase were prepared for electron microscopy in a thin film of vitrified buffer. This was then devitrified as before so that the catalase crystals were embedded in a thin film of cubic ice. A typical specimen area is shown in Fig. 2b. In areas where the film is thicker, individual ice crystallites overlap with the catalase crystals (Fig. 2a). Electron diffraction patterns of catalase crystals on the same grid before and after devitrification extended to 2 A resolution (results not shown), demonstrating that the formation of ice crystals during devitrification did not disrupt high-resolution detail. Low-dose images of thin
NL Cyrklaff, F¢~Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
145
Fig. 1. Film of unsupported cubic ice_ (a) Low-magnification image showing the characteristic granular appearance. (b) High-magnification image (electron-optical magnification: 250k x ) of a single crystallite measuring about 1000 A, across, showing lattice fringes (111) of cubic ice (1¢ = 3.66 ,~). The inset shows the corresponding optical diffraction pattern with I c (444) = 0.92 A (arrow). Cubic ice was produced by controlled devitrification of water in the electron microscope, and imaged with a dose of approximately 100 e ,~-2. Space bar (a) 2 ~zm and (b) 100 A,.
146
M. Cvrklc(~ W. KtThlbrandt / UItramicroscopy 55 (1994) 141 153
12,11 )
12~8
Fig. 2. (a) Thin 3D catalase crystal embedded in ice. (b) High-magnification image showing the catalase lattice, recorded with 10 e 2 at magnification 60k × . (c) Optical diffraction pattern of a catalase crystal similar to the one shown in (a). (d) Part of the calculated, background-corrected Fourier transform of the same image, showing spots on the reciprocal lattice beyond the 8th order, extending to 5.4 A. The cell dimensions of beef liver catalase crystals are a 69 A, b = 173.5 ,~ [31]. Space bars (a) I # m and (b) 500 ,~
M. Cyrklaff, ~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
catalase crystals were examined by optical diffraction and showed reflections to 5.4 A (Fig. 2c and 2d). Images of ice-embedded specimens gave somewhat sharper reflections and the success rate of recording high-resolution images was higher than with vitrified specimens.
147
OD 1.5-~
i
i
I
i
i
I
i
0,02
0
1.0
3.3. Tobacco mosaic c'irus As a second test specimen for high-resolution imaging we used tobacco mosaic virus in vitrified buffer and cubic ice.
0.5
3.3.1. Electron diffraction of TMV In concentrated suspension, T M V particles tend to align, making rafts measuring up to 6 / x m in diameter. Occasionally such rafts are suffi-
0 0 04
I
0.02
I
I
0.04
-1
~,
Fig. 4. Intensity profile of the meridional range of the 6th layer line in the electron diffraction pattern of TMV shown in Fig. 3. The modulation of the intensity shows that the TMV particles were well-aligned.
ciently well ordered for high-resolution electron diffraction [22]. The layer line intensities recorded in the diffraction pattern thus represent an average over many hundreds of particles. An example of an electron diffraction pattern of T M V in vitrified buffer is shown in Fig. 3. The 31st layer line is visible at the meridian, corresponding to a resolution of 2.3 A in this direction. In the equatorial direction, reflections are more diffuse and extend to roughly 5 ,~. The intensity profile of the 6th layer line, at 11.5 ,& resolution at the meridian (Fig. 4), indicates that the particles were well oriented within the raft. Scaled values of maximal, background-corrected electron diffraction intensities on each layer line are listed in Table 1. The ratio of maximal intensities on the 3rd layer line at 23.0 ,~ resolution and the 6th layer line was 0.56.
Fig. 3. Electron diffraction pattern of a TMV raft_ Camera length 1500 mm, dose below 1 e ~ - 2 . The lower half shows the low-resolution 3rd and 6th layer lines, as used for calculating the intensity ratio of them. Rings are due to subtraction of rotationally averaged background_
3.3.2_ Images of TMV Images of T M V were recorded in vitrified and devitrified buffer. In order to optimize contrast in the 7 - 1 2 A resolution range, images were taken at an underfocus of approximately 1500 to 2000 •~. At this defocus, with an accelerating voltage of 200 kV and C~ = 1.9 mm, the first C T F minimum occurs at a spatial frequency of (6.0 ~,) 1 to (7.2 ~,)-t. Since low-resolution contrast is weak under
h/L Cyrklaffl W Kiihlbrandt / Ultramicroscopy 55 (1994) 141 153
148
these conditions, the virus particles were virtually invisible by eye on the electron micrographs. The rods could, however, be located precisely by optical diffraction, scanning the negatives with a rectangular aperture with a width corresponding to that of the T M V particles. Of the images which were properly focused, a total of about 150 of devitrified and about 100 of vitrified T M V specimens were evaluated by optical diffraction. The 6th layer line of the T M V diffraction pattern, corresponding to a resolution of 11.5 A, was detectable in about 55% of devitrifled, and in about 40% of vitrified samples. The 3rd layer line was comparatively weak, due to the contrast transfer function (CTF) at this defocus. T M V particles showing detail beyond the 8th layer line were found only on devitrified specimens. The best ones of these showed the 9th layer line, at a resolution of 7.7 ,&. In one instance, the 12th layer line, at approximately 5.75 ,~, was visible by optical diffraction (Fig. 5), using an aperture corresponding to the width of three parallel T M V rods. With this larger aperture, the observed layer line intensities were stronger by a factor of roughly 5 compared to a single T M V particle. In the Fourier transform of this area, the 10th, l l t h and 12th layer lines had peak intensities above background of, respectively, 8, 9 and 14 after scaling to the 6th layer line as in Table 2.
Six separate areas on the four best images (three of devitrified, one of a vitrified specimen) were digitized on a microdensitometer after masking the area on the negative, with the long axis of the particles parallel to the X-scan direction. The Fourier transforms of each digitized area were then examined for maximum intensities along each layer line. Results are shown in Table 2_
3.3.3. Assessment of TMV image quality
o
The electron diffraction intensities of T M V provide an objective criterion for assessing the quality of images. In an ideal image, the intensities in the Fourier transform should be identical to those measured by electron diffraction. In reality, however, the intensities in particular of highorder terms are severely reduced by specimen-related effects, such as inelastic scattering, radiation damage, sample movements and charging. In addition, the molecular transform is modulated by the contrast transfer function (CTF) of the electron microscope. If the CTF is known, then corrected structure factor intensities can be calculated by multiplying each image intensity with the inverse of the CTF for a particular spatial frequency. In practice, the CTF is determined from the positions of the Thon-rings in the optical diffraction pattern, i.e_ the spatial frequencies
Table 1 Structure factor intensities in the electron diffraction pattern of cryo-hydrated TMV rafts Layer line
1
2
3
4
5
6
7
8
9
10
11
12
Intensity
n.d.
n.d.
56.3
20.1
26.7
100.0
42.1
17.9
17.8
14.5
13.2
18.8
Layer line
13
14
15
16
17
18
19
20
21
22
23
24
Intensity
11.4
11.6
35.0
13.8
11.4
21.5
11.2
12.3
13.0
14.6
11.9
13.3
Layer line
25
26
27
28
29
31)
31
Intensity
11.0
11.4
11.3
11.0
10.8
11.1
9.6
Background-corrected structure factor intensities of the TMV electron diffraction pattern. The maximum intensity for each layer line is shown, scaled to the 6th layer line. The first and second layer lines were not visible due to the high background close to the origin.
M. Cyrklaff, I~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
This is less and less likely at increasing magnification. However, the best image of T M V in vitrified buffer (no. 3296) shows an area of carbon film which allowed us to determine the first minimum of the CTF and thus to correct the observed intensities. The CTF was calculated assuming 7% amplitude contrast as found for unstained weak-phase, weak-amplitude specimens imaged in the microscope with 100 kV electrons [23]. At an accelerating voltage of 200 kV, the amplitude contrast would be somewhat lower, and this would slightly increase the correction factor. The intensities were not corrected for the envelope function which is difficult to estimate. The envelope would decrease the high-resolution intensities and therefore lower the correction factor. The intensity ratios of the 3rd versus 6th layer lines for three different particles in this image ranged from 2.9 to 4.9 (Table 2). This is much worse than the ideal value of 0.56 obtained by electron diffraction, indicating a major loss of contrast at a resolution of 11.5 A and above. However, these values compare favorably with others reported previously for T M V in vitrified suspension [12,14,24]. Only one of the 3 best images of T M V in cubic ice contained any carbon film, so that the CTFcorrected structure factor intensities could not be determined exactly for all of them. Since we aimed to record all images at defocus between 1500 and 2000 A, we can assume, however, that the C T F is similar for all images and close to maximal for the 6th layer line at 11.5 ,~ resolution. Making this assumption, we obtained intensity ratios of the 3rd versus the 6th layer line of 2.3 (_+0.56) for a single T M V particle. For the Fourier transform of three parallel T M V rods in the same image area, this ratio improved to 1.8 (_+0.3) as a result of the increased signal/noise ratio. Cubic ice therefore seems to be preferable to vitrified water as a medium for high-resolution electron cryo-microscopy. Table 2 also shows the ratio of the strongest diffracted intensity (on the 6th layer line) to the intensity at the origin, as a measure of absolute contrast. For the images, these ratios range from 2 x 10 -.7 to 3.5 x 10 -~, which is roughly three orders of magnitude lower than the ratio estio
Fig. 5. Optical diffraction pattern of three parallel T M V rods, 0.2 Izrn long. The arrow points to the 12th layer line, corresponding to a resolution of 5 75 ,~. The layer lines are appearing on one side of the meridian only. This is characteristic for one-sided helices and may indicate partial distortion of the particles for example by protrusion through the water surface and partial dehydration.
for which the contrast is zero. The contrast of biological specimens in vitreous water or cubic ice is too low to give rise to detectable Thon rings. However, in favorable circumstances, a good estimate of the C T F can be obtained if the imaged area includes some carbon film.
149
n.d.
Electron diJ]kaction
n.d.
43.2 10.0 47.2
12.3 25.3 55.2
0.063
2
56.3
90.5 48.0 45.6
78.8 61.8 103.0
0.185
3
2/).1
27.2 9.4 25.6
9.0 19.3 24.5
0.397
4
26.7
57.5 6.1 49.4
11.2 17.2 28.2
0.722
5
100.0
100.0 100.0 100.0
100.0 100.0 100.0
0.884
6
42.1
40.2 7.0 20.3
6.7 19.8 23.5
0.640
7
17.9
29.6 3.9 19.6
10.0 13.8 28.8
0.137
8
17.8
19.4 5.0
-
0.032
9
0.56
0.91 0.48 0.46
0.79 0.62 1.03
Ratio 3rd/6th
4.35(+ / 2.29(+/ 2.19
3.77 2.96 4.92
Ratio 3rd/6th (CTF) 2 corrected
0.56) * 0.30)*
5.2x 10 4
4.3 x 10 s 2.2x10 7 3.5 x l0 s
2.0x10 7 9.0x 10 ~ 6.6x 10 -8
Origin/6th layer line intensity
Background-corrected intensities were scaled to the 6th layer line intensity before CTF correction. The first minimum of the CTF was obtained by optical diffraction of carbon film in image #3296, and calculated for 200 kV accelerating voltage, with the objective lens spherical aberration value equal to 1.9 mm, and assuming 7% amplitude contrast [23], without including an envelope decay function. This CTF was used to correct the intensities for this image, as well as for the ice-embedded images indicated by *. Estimated error margins for the correction are _+ 13%, due to the uncertainty of defocus. The CTF for image #2739 was measured in the same way and found to be very similar. Selected electron diffraction intensities from Table 1 are given in the bottom row for comparison. The ratio of intensity of the 6th layer line to the intensity at the origin was derived from image transforms of TMV. The ratio for electron diffraction was calculated from the number of electrons calculated from the total integrated intensity above background of the 6th layer line to the estimated total number of electrons used to record the diffraction pattern.
43.8 8.5 35.2
12.5 21.1 19.8
0.014
(CTF) 2
1
Layer lines
2908 2912 2739
Ice
3296 1 3296 2 3296 3
Vitrified
Image number
Table 2 Structure factor intensities in the computed Fourier transforms of TMV in cubic ice and vitrified water
',.a t~a
I
4k
©
M. Cyrklaff 144 Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
mated from an electron diffraction pattern (5.2 × 10-4). However, the ratio is 5 times higher for image transforms of three parallel T M V rods (1.2 × 10-6), due to the higher signal/noise ratio.
4. Discussion
The frozen hydrated preparation method should be ideal for preserving high-resolution detail of any biological specimen. However, it is striking that near-atomic image resolution has, so far, been achieved more reliably with specimens in glucose and tannin, rather than in vitrified buffer. This indicates that the problems associated with high-resolution imaging are at least in part due to the water support. One of these problems is caused by small movements of the specimen and the supporting film as they absorb energy from the electron beam. It may be expected that a film composed of small, tightly interlaced crystals is mechanically more rigid and hence less prone to move under the beam. The results presented here demonstrate that this is indeed the case. The fact that electron micrographs of the cubic-ice lattice diffracting to 1 - 2 / ~ in the laser b e a m can be recorded routinely indicates clearly that it is a rigid, mechanically stable support film. The crystal lattice makes it easy to check whether or not specimen movement has occurred while an image was recorded and to test the performance of the electron microscope and of the specimen holder at conditions used for cryo-microscopy. It is possible that small specimen movements and the build-up of charges occur mainly in the initial period of exposure and that the specimen stabilizes after about 10 e ~ - 2 . This may account for the high resolution of the ice-lattice images which were recorded with a dose of 100 e ,&-2 which is more than 10 times higher than what can be used in biological electron cryo-microscopy at liquid nitrogen temperature. Nevertheless, the success rate of recording 11.5 A images of T M V particles indicates the increased mechanical stability of cubic ice compared to vitrified buffer. Ice-crystal formation can cause severe damage
151
to biological samples, primarily because the growing ice crystals exclude solute molecules and ions, thus causing osmotic stress in the remaining volume. The disruption of the hydration layer around macromolecules could be another cause of damage. Because freezing is a strongly exothermic process, it takes place generally at a temperature only slightly below the freezing point of 0°C. Freezing damage is avoided when water is vitrified, namely when it is cooled so rapidly that it has no time to start forming ice crystals before the viscosity becomes so high that any movements are practically blocked [25]. In the present work, ice-crystal formation takes place under conditions which are very different from the usual freezing around 0°C in that water molecules immobilized in the vitrified state are rewarmcd to a temperature where crystallization occurs. Apparently, crystallization of water under these conditions involves only minimal molecular displacements, and we have shown that biological macromolecules are unaffected by this process. It has been observed before that some crystalline biological specimens are well preserved even in presence of large ice crystals [26]. This was thought to be due to the fact that the crystals form from amorphous water [27], consistent with the results presented here_ Taylor and Glaeser [28] found that thin crystals of catalase diffract to 2.8 A even after freezing in liquid nitrogen which results in the formation of large ice crystals. Catalase crystals have a high protein packing density and presumably contain little liquid water which may explain why they are less sensitive to freezing damage. However, it is well known that freezing in liquid nitrogen damages single particles and other specimens with a high water content [17]. The procedure applied by us requires careful control of the specimen temperature_ If this rises above -90°C, where water begins to sublimate rapidly, there is a risk of freeze-drying the specimen. Our images of T M V and catalase show that this has not occurred since they are clearly embedded in a continuous film of microcrystalline buffer (Fig. 2a). The T M V images were recorded with an objective lens defocus of 1500 to 2000 A and thus much closer to focus than in other studies
152
M. Cyrklaff V~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141 153
[12,14,24]. At this defocus, the CTF maximum is close to the 6th layer line (in image 3296 located below the first maximum with a value of 0.884), whereas the 9th and higher layer lines lie beyond the first minimum. Low-resolution frequencies are de-emphasized so that the typical virus features (a rod of 180 A width, with 23 A striations) are not visible by eye of the micrographs. However, single helical rods of T M V can be easily localized by optical diffraction. In this way, structural information in the l 1.5 to 7.5 ,~ resolution range can be recorded with single T M V particles. Clearly, averaging over several T M V images of this quality w o u l d greatly i m p r o v e the signal/noise ratio. Following a suggestion by Henderson [14], we used the ratio of image intensities of the 3rd versus the 6th layer line to assess the quality of an image. This ratio compares information about molecular envelope (3rd layer line) with that about the arrangements of secondary structure within the protein (the 6th layer line and higher). This ratio was calculated both for images and for the electron diffraction patterns_ We find that for the electron diffraction patterns the ratio of the 3rd versus the 6th layer line is higher by a factor of 1.5 compared to X-ray diffraction, exactly as predicted by Henderson [14]. This appears to be due to the increased low-resolution contrast of T M V in ice (which has a density of 0.92 g ml J) compared to T M V in water. In the computed transforms of T M V images, corrected for the contrast transfer function, the ratios of the 3rd to 6th layer lines intensities are on average six times higher than the reference, whereas values reported by Henderson [14] for the T M V images collected in various electron microscopes at considerably higher defocus are between 11 and 35. This is partially due to the fact that our images were closer to focus. We realize, however, that images in which the particles are not visible by eye will be useful only if their position can be detected in some other way, e.g. by optical diffraction, or by recording a second image at higher defocus. We hope that these studies will contribute towards imaging fully hydrated, crystalline and non-crystalline biological specimens at high reso-
lution. At present, the absolute contrast of our best image of a single particle is about 1000 times worse than in a perfect image, which suggests that very large improvements can be expected, once the remaining problems have been identified and overcome. This should be possible in the near future, both by improvements in instrumentation and in specimen preparation. Instrumental improvements include higher acceleration voltage, lower specimen temperature, the use of energy filters [29], and objective lens correctors [30]. Improvements in specimen preparation may include evaporating a conducting layer onto one or both ice surfaces to reduce specimen charging and movement.
5. Conclusions (1) We have shown that thin films of cubic ice, produced by controlled devitrification of vitreous water, provide a rigid and stable support suitable for high-resolution imaging of biological macromolecules. The cubic-ice film is stable to within 1 A. (2) The ice lattice can be used as a convenient test object to assess the performance of the electron microscope and the specimen holder used for cryo-imaging. (3) Biological macromolecules embedded in cubic ice were imaged at higher resolution and with better signal/noise ratio than comparablc specimens embedded in vitreous water. (4) Thin crystals of catalase embedded in vitrified medium or in cubic-ice-diffracted electrons to 2 A, indicating that the specimen was not damaged by the formation of ice crystals during devitrification. (5) Electron diffraction patterns of oriented T M V rafts provided a reference for assessing the quality of single-particle images. Image intensities between 23 and 11.5 A resolution decay roughly 6 times more rapidly than electron diffraction intensities, indicating that large improvements in image quality are still required. o
M. Cyrklaff, l~ Kiihlbrandt / Ultramicroscopy 55 (1994) 141-153
Acknowledgements We gratefully acknowledge Richard Henderson a n d J a q u e s D u b o c h e t for critical c o m m e n t s , and Kevin Leonard for helpful suggestions. M.C. specially thanks Bjcrn Johansen and Norbert Roos for support and valuable discussions during his visits to O s l o U n i v e r s i t y .
References [1] R. Henderson, J.M. Baldwin, K.H. Downing, J. Lepault and F. Zemlin, Uhramicroscopy 19 (1986) 147. [2] J.M. Baldwin, R. Henderson, E. Beckmann and F Zemlin, J. Mol. Biol. 202 (1988) 585 [3] B.K. Jap, P.J. Walian and K. Gehring, Nature 350 (1991) 167. [4] H.J. Sass, G. Bfildt, E. Beckmann, F. Zemlin, M. van Hill, E. Zeitler, J.P. Rosenbusch, D.L. Dorset and A. Massalski, J. Mol. Biol. 209 (1989) 171 [5] W. Kfihlbrandt and K.H_ Downing, J. Mol. Biol. 207 (1989) 823. [6] DN. Wang and W. Kiihlbrandt, J Mol. Biol. 217 (1991) 691. [7] R. Henderson, J.M_ Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann and K.H. Downing, J. Mol. Biol. 213 (1990) 899. [8] W. Kiihlbrandt, D.N. Wang and Y. Fujiyoshi, Nature 367 (1994) 614. [9] D.L. Stokes and N.M. Green, J. Mol. Biol. 213 (1990) 529. [10] J. Brink, W. Chiu and M. Dougherty, Ultramicroscopy 46 (1992) 229.
153
[11] R. Grant, M.F. Schmidt and W. Chiu, J. Mol. Biol. 217 (1991) 551. [12] T.-W. Jeng, R.A. Crowther, G. Stubbs and W. Chiu, J_ Mol. Biol. 205 (1989) 251. [13] N. Unwin, J. Mol. Biol. 229 (1993) 1101. [14] R. Henderson, Ultramicroscopy 46 (1992) 1. [15] A. Fukami and K. Adachi, J. Electron Microsc. 14 (1965) 112. [16] N.G. Wrigley. J. Ultrastruct. Res. 24 (1968) 454. [17] J. Dubochet, M. Adrian, J.-J. Chang, J.-C. Homo, J. Lepault, A.W. McDowall and P. Schultz, Q. Rev. Biophys. 21 (1988) 129. [18] R. Henderson, C. Raeburn and G. Vigers, Ultramicroscopy 35 (1991) 45. [19] J.-C. Homo, J. Microscopy 136 (1984) 337. [20] W. Ki~hlbrandt, J. Mol. Biol_ 202 (1988) 849. [21] M. Adrian, J. Dubochet and A.W. McDowall, Nature 308 (1984) 32. [22] T.-W. Jeng and W. Chiu, Ultramicroscopy 23 (1987) 61. [23] C. Toyoshima and N. Unwin, Ultramicroscopy 25 (1988) 279. [24] R.R. Schr6der, W. Hofmann and J.-F. Menetret, J. Struct. Biol. 105 (1990) 28. [25] F. Franks, Properties of aqueous solutions at subzero temperatures, in: Water: A Comprehensive Treatise. Water and Aqueous Solutions at Subzero Temperatures, Ed. F. Franks (Plenum, New York, 1982) p. 215. [26] R.A. Milligan, A. Brisson and P.N.T. Unwin, Ultramicroscopy 13 (1984) 1. [27] J. Dubochet, K. Richter, H.-V. Roy and A.W. McDowall, Scanning Microsc. Suppl. 5 (1991) 11. [28] K.A. Taylor and R.M. Glaeser, J. Ultrastruct. Res. 55 (1976) 448. [29] R.R. Schr6der, J. Microscopy 166 (1992) 389. [30] H. Rose, Optik 85 (1990) 19. [31] P.N.T. Unwin, J. Mol. Biol. (1975) 235.