Critical Review. Viruses and the Development of Quantitative Biological Electron Microscopy*

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1 IUBMB Life, 56(5): , May 2004 Critical Review Viruses and the Development of Quantitative Biological Electron Microscopy* R. A. Crowther MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Summary The electron microscope has become an important tool for determining the structure of biological materials of all kinds. Many technical advances in specimen preparation and in sophisticated methods of image analysis, initially based on optical systems but latterly on computer processing, have contributed to the development of the subject. Viruses of various kinds have often provided a convenient and appropriate test specimen. This paper describes the major technical advances and shows how viruses have had an important role in most of the developments. IUBMB Life, 56: , 2004 Keywords Electron microscopy; image processing; three-dimensional reconstruction; virus structure INTRODUCTION From its invention in the 1930s the electron microscope gave the potential for visualizing biological particles, such as viruses, that were too small to be seen by light microscopy. Indeed, some of the first electron images of biological material were of bacterial viruses or bacteriophages (1). However, many technical developments were needed before the full power of the approach could be realized. Viruses, being intrinsically interesting, readily purified and possessed of various kinds of symmetry, provided a key test subject for many of these developments, and in turn the electron microscope revealed many important aspects of virus structure. The part played by Fellows of The Royal Society in the broad historical development of microscopy has been reviewed Address correspondence to: R. A. Crowther, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. rac1@mrc-lmb.cam.ac.uk *This article first appeared in Notes and Records of the Royal Society of London Crowther, R.A. 2004, 58, and is republished by kind permission of the Royal Society and the author. previously (2). Many of those involved in the later developments in biological electron microscopy, to be described in more detail here, are or were Fellows. 1 Biological samples are delicate, hydrated and composed of atoms of low atomic number. This means that they are easily destroyed by exposure to the electron beam, that it is difficult to introduce them into the high vacuum of the microscope in a native state and that when they are photographed they give rise to images with very low contrast. As outlined below, these difficulties were partly overcome by using heavy metals to coat or stain the particles, thus producing a relatively stable, radiation-resistant and contrasty specimen. However, the information recovered in this way about the specimen is limited, and methods have been developed more recently for examining unstained, native specimens. Whatever the method of preparation, the resulting micrographs can be difficult to interpret directly in detail, because features at different levels in the three-dimensional specimen become superimposed in the two-dimensional image recorded on the micrograph. In effect, the microscope produces an image of the specimen projected in the direction of the beam. In general it is not possible to deduce much about the detailed three-dimensional structure of the specimen from a single image of this kind, although as we shall see for some symmetrical specimens quite a lot can be learned. The geometrical symmetry possessed by many biological specimens has often been central to the analytical approaches used for extracting reliable information from the micrographs. The repetitive nature of the image of a symmetrical structure gives the chance of averaging the features in the image to reduce the effects of granularity of stain, of contributions to the image from the specimen support film and, for low-dose images, the fluctua- 1 Of those mentioned in the paper as contributing to developments in quantitative biological electron microscopy, the following are or were Fellows of the Royal Society: Dr. S. Brenner, Dr R.A. Crowther, Dr J.T. Finch, Dr R. Henderson, Dr H.E. Huxley, Dr A. Klug, Dr P.N.T. Unwin and the late Dr R. Markham. ISSN print/issn online # 2004 Royal Society DOI: /

2 240 CROWTHER tions in the numbers of electrons recorded with the limited exposure possible. This kind of processing, which reinforces the meaningful parts of the signal and reduces the noise, started with relatively simple optical methods but now depends heavily on the sophisticated use of computers to analyse the micrographs and produce detailed three-dimensional maps. This paper gives a personal and selective view of some of the key developments in the story, concentrating on the technical developments themselves rather than the detailed results. DEVELOPMENT OF NEGATIVE STAINING The first methods of contrast enhancement involved either shadowing the specimen with a beam of heavy-metal atoms or positively staining the specimen with a concentrated solution of a heavy-metal salt and washing away the excess salt. In the former case a layer of metal atoms was deposited on the particle, which also cast a shadow; in the latter the metal atoms reacted chemically with the specimen. Both approaches gave information about the size and general shape of virus particles but neither provided much detail. In the course of this kind of experiment Hall (3) noted the following in insufficiently washed specimens of stained tomato bushy stunt virus: The lightly stained particles are embedded in the dried reagent and therefore appear as holes because of their relatively low scattering power. Although the effect is the opposite to what is usually sought by the use of electron stains, the visibility of the particles of low scattering power can be enhanced as well, if not better, by surrounding them with dense material rather than impregnating them with dense material. high resolution electron microscopy of viruses. The virus preparation was mixed with 1% phosphotungstic acid, then sprayed onto a thin carbon film on the microscope grid and allowed to dry. In this way the virus particles became embedded in a thin coat of stain, which for the first time allowed details of molecular structure on the virus surface to be revealed (Fig. 1). Negative staining, a term taken from earlier light microscopy (6), became the standard way of looking at particulate material, especially viruses, and remains in use today as a simple way of preparing robust specimens for microscopy. The main contribution to the contrast variation in the recorded micrograph comes from the stain coat, and holes and modulations in this coat are assumed to be biological material, giving rise to the use of the word negative. It is as though one were making an X- ray radiograph of a plaster-cast of the virus (7). The fidelity with which details of the molecular packing on the surface of the particle are revealed by negative staining is remarkable, but the definition of internal molecular structure is very limited. INTRODUCING VIRUS MORPHOLOGY AND MODEL BUILDING Simple viruses have one of two morphologies, rod-shaped or spherical. This distinction emerged from the earliest electron micrographs. Crick and Watson (9) presented a In the following year, in a paper on the structure of tobacco mosaic virus (TMV), Hugh Huxley (4) visualized the central hole along the axis of the rod-shaped particle and noted: It was sometimes apparent that the excess stain had not been completely removed by washing before the preparation was allowed to dry. In these areas, the particles became outlined by stain in a very distinctive manner.... Thus if a TMV particle is allowed to dry in a pool of dilute salt solution, the salt will tend to be deposited around the surface of the particle, and if the particle has an inner surface which is accessible to salt, the salt will be deposited there too. He concluded: The outlining technique would appear to be quite a useful one for this type of specimen, particularly as it is so simple and gives excellent contrast and resolution. This approach was formalized and named by Brenner and Horne (5) in a paper entitled A negative staining method for Figure 1. Montage of negatively stained virus particles. (a) Tobacco mosaic virus. (b) Disc of tobacco mosaic virus coat protein. (c) Human adenovirus with (d) a model of the structure (8). (e) Tomato bushy stunt virus. (f) Bacteriophage T4 and (g) the baseplate of T4. The various images are at approximately the same magnification and the scale bar in (e) represents 20 nm. Micrographs (a), (b) and (e) were kindly supplied by Dr J. T. Finch. Micrographs (c) and (d) reprinted with permission from Elsevier.

3 VIRUSES AND DEVELOPMENT OF ELECTRON MICROSCOPY 241 powerful hypothesis, based on genetic economy and protein interactions, to suggest that in simple viruses the coats would be made from multiple copies of a relatively small protein, symmetrically arranged. This would mean that every subunit made exactly equivalent interactions with its neighbours. For rod-shaped viruses this would imply helical symmetry, whereas for spherical or isometric viruses the symmetry would be cubic. With a helix an arbitrarily large number of subunits can be incorporated, but with a closed shell the largest number of subunits that can be accommodated is 60, when the symmetry is icosahedral. With only 60 subunits it might not be possible to make a sufficiently large coat to contain the genetic material. Caspar and Klug (10) proposed an extension of this idea, so that certain multiples of 60 subunits could be incorporated to make an icosahedral shell but at the expense of making the interactions between the subunits no longer exactly equivalent. This formalism provided a powerful impetus to the interpretation of the features of virus structure being increasingly revealed at that time by electron microscopy and X-ray diffraction. Images of some of the viruses mentioned in this paper are shown in Fig. 1. The prototype rod-shaped virus is TMV. The coat protein on its own assembles into discs with 17-fold rotational symmetry. Examples of spherical viruses are provided by tomato bushy stunt virus and human adenovirus, with the model of the latter demonstrating the symmetry of the icosahedron. Bacteriophage T4 is more complicated and consists of an elongated head, with caps based on icosahedral symmetry, a helical rod-shaped tail and a terminal hexagonal baseplate. The symmetries in the particles are exploited by the different image-processing procedures to be described. The ability to record molecular details of virus structure immediately raised the question of the structural interpretation of the features seen in the images. Initial attempts at understanding viruses and their electron microscope images were based on physical model building. An early example of a pingpong ball model of adenovirus is shown in Fig. 1d (8). In this case the structure and the direction of view are such that features in the front and back of the particle exactly superimpose, so that the surface appearance of the model agrees broadly with the projected view in the micrograph (Fig. 1c). Generally this is not the case. By making models out of stick-like elements it was possible to create superpositions from shadow patterns cast by the model (Fig. 2c) (11). Although somewhat more realistic, the shadow pattern can only be black or white and so does not represent a true projection. By this time computer film plotters were becoming available, so much more realistic projections could be produced (Fig. 2d) (12). However, model building involved trial and error and there was no guarantee that any model could be invented to explain all the features in the images. More direct and quantitative methods were needed, and their development had already started. FIRST ATTEMPTS AT IMAGE AVERAGING As pointed out above, the image of the biological object is degraded by extraneous noise arising from the granularity of the stain, contributions from the supporting carbon film and distortions of the sample. If the object is made from repeated units arranged in a symmetrical way, it is possible to enhance the signal and reduce the noise by superimposing the different copies of the unit. The first proposal for doing this was made by Markham and colleagues (13), who sought to exploit the rotational symmetry of various viruses and their subassemblies by using a photographic superposition method. This was inspired in part by an earlier demographic study by Galton (14), who tried to extract the typical facial features of different groups of people, including criminals, by the averaging of photographic portraits. In Markham s method a photographic enlarger is used to make multiple superimposed prints of the micrograph, the photographic paper being rotated by 360/n degrees between exposures, where n is the order of rotational symmetry being tested. By trying different values of n and choosing the value that gives the most visually convincing image, the most likely order of rotational symmetry is inferred and at the same time an averaged image with improved signal : noise ratio is produced. Unfortunately the principal result in the paper, which seemed to show that discs of TMV coat protein had 16-fold rotational symmetry, was incorrect. This was nevertheless a pioneering attempt at the image processing of electron micrographs. In the following year Markham and his colleagues (15) described a linear integrator, again based on multiple photographic exposures of the micrograph, but this time with linear translation rather than rotation between the exposures. In this way linearly periodic features in the micrograph could be detected and enhanced. TMV again provided the test specimen, and the results with the rods of the stacked-disc form of the coat protein were particularly convincing. The operation both determined the period of the dominantly repeating features in the image and simultaneously created an enhanced image. However, the weakness of the technique was that it had to be based on trial and error, and the judgement of what was significant, as with rotational superposition, had to be made subjectively by looking at the differently averaged images. OPTICAL DIFFRACTION AND FILTERING A completely different approach to analyzing the periodicities present in electron micrographs was taken by Klug and Berger (16). In their words, Clearly what is required is a decomposition of the image into its components i.e. a complete Fourier analysis. The Fraunhofer diffraction pattern of the image would automatically provide just such an analysis. By using the image on the micrograph as a light optical diffraction grating in an optical diffractometer (Fig. 3), a device already introduced by Lipson, Taylor and co-workers

4 242 CROWTHER Figure 2. Examples of model building to interpret images of human wart virus (11, 12). (a, b) Images of negatively stained particles. (c) Shadowgraph and (d) computer simulation. All views are down the 3-fold symmetry axis of the icosahedron. Reprinted with permission from Elsevier. Figure 3. Schematic diagram of the optical system used for optical diffraction and filtering (19). The electron micrograph forms the subject, and the diffraction pattern is formed in the diffraction plane; this is also where the mask is inserted to create a filtered image, which is then observed in the image plane. Reprinted with permission from Nature Publishing Group. for the interpretation of X-ray diffraction patterns of crystals, the different periodicities present in the micrograph give rise to beams diffracted at different angles. Recording the diffraction pattern captures the Fourier transform of the image, so that spots corresponding to dominant periodicities are immediately apparent. TMV was again used as a test specimen and in an accompanying paper Finch, Klug and Stretton (17) analyzed an aberrant tubular form of the T4 bacteriophage head, known as a polyhead (an example is shown in Fig. 4) (18). The importance of the paper by Klug and Berger was that it introduced the explicit use of the Fourier transform in analysing micrographs, a mathematical technique that subsequently became indispensable. In this context the Fourier

5 VIRUSES AND DEVELOPMENT OF ELECTRON MICROSCOPY 243 Figure 4. An example of optical filtering (18). The specimen (a) is a mutant tubular form (polyhead) of the T4 phage head. (b) Its optical transform, with rings drawn around the set of spots arising by diffraction from one side of the tube. The unringed spots arise from the other side of the tube. (c, d) Filtered images of the two sides of the tube, obtained by recombining one or other of the two sets of spots. The arrangement of protein subunits in a regular array of hexagonal rings is clear. Reprinted with permission from Elsevier. transform was crucial because it gave an objective and simultaneous view of the strengths of all the spatial frequencies present in a micrograph, rather than the subjective sampling of individual periodicities given by Markham superposition. The other important notion addressed in the paper was to understand the mechanism of negative staining and in particular the idea that contrast in the image arises by superposition of stain deposited on both the top and bottom of the specimen (that is, the sides nearest and furthest from the carbon support). Thus, negative staining generally gives a cast of the particle rather than a footprint, as had been believed by some people. In extended tubular structures such TMV or T4 polyhead, the periodic features on the near and far sides give rise to spatially separated diffraction spots that can be indexed; in other words, the contributions can be assigned systematically to one side or the other (Fig. 4b). If one set of spots is consistently stronger than equivalent spots in the other, this shows that the two sides are not equally stained and are contributing independently to the total image contrast, which is therefore arising by superposition. The specimens might be well ordered but their images can still look complicated and not be immediately interpretable, because of the superposition. Once optical diffraction had been established, a natural next step for extended periodic specimens, in which the diffraction spots corresponding to the two sides of the specimen were spatially separated, was to manipulate the diffraction pattern by introducing a mask or filter to remove one set of spots and to recombine the other set to form an image of a single side of the specimen (Fig. 4). This process was realized by Klug and DeRosier (19) in their description of a set-up for optical filtering (Fig. 3), based on a modification of the Taylor Lipson diffractometer. Besides separating the two sides, the mask also controls the degree of averaging in the filtered image, with smaller holes giving a greater range of averaging of the periodic features. TMV again, together with a helical bacteriophage tail, provided the experimental subjects. By this approach the steps of the analysis of the original image (by optical diffraction) and the synthesis of an averaged image of a single side (by optical filtering) were separated, and thus a large part of the subjectivity in the Markham technique was removed. It was of course still necessary to analyze many examples of any given specimen, to see that the indexing and filtering were reproducible and consistent. Later an analytical technique for rotational filtering was also developed (20). Unlike translational symmetry, in which the diffracted beams produce separated pointlike features in the transform, in rotational symmetry the different rotational components overlap one another in the transform. To separate them it was necessary to use computational methods to decompose the image, or equivalently its transform, numerically into a complete set of harmonic components. The strengths of these components could then be plotted as a rotational power spectrum, analogous to the peaks in the optical diffraction pattern of a translationally periodic image. It was easy to test different positions for the best rotational centre and to see whether a particular rotational symmetry were dominant. The harmonics consistent with this symmetry could then be recombined to create a rotationally filtered image. As with optical diffraction and filtering, the analysis and synthesis steps were thus separated and the whole approach was more quantitative and less subjective than Markham rotational superposition. Discs of TMV coat protein and the baseplate of T4 bacteriophage were used as test specimens. For the former the rotational symmetry was shown correctly to be 17-fold, unlike the 16-fold given by Markham superposition, indicating the greater reliability of the analytical approach. For the latter an extensive study of mutants of the T4 baseplate gave the first information about the locations of particular proteins in the baseplate (Fig. 5) (21). THREE-DIMENSIONAL RECONSTRUCTION The next and major advance was prompted by the fact that even when the optical filtering approach had been used on something like the helical tail of T4 bacteriophage, to separate near and far sides, the image of one side was still a superposition of features at different cylindrical radii (Fig. 6). This prompted DeRosier and Klug (22) to propose a general method for Reconstruction of three dimensional structures from electron micrographs. Although the method was here applied to the rather special case of a helically symmetrical specimen, the general applicability of the approach was emphasized. They wrote:

6 244 CROWTHER Figure 5. Rotationally filtered images of T4 phage baseplates (21), showing (a) the hexagonal form associated with an extended tail and (b) the star-shaped form that arises after tail contraction during infection. Details of the molecular anatomy are much clearer than in the unprocessed images (for example Fig. 1g). Reprinted with permission from Elsevier. Figure 6. T4 phage tail showing (a) the original image, (b) its optical diffraction pattern and (c) the filtered image of one side of the tail (22). It is clear from the different slopes of the two sets of helices seen in (c) that there is still superposition of features at different cylindrical radii. Reprinted with permission from Nature Publishing Group. Our method starts from the obvious premise that more than one view is generally needed to see an object in three dimensions. We determine first the number of views required for reconstructing an object to a given degree of resolution and find a systematic way of obtaining these views. The electron microscope images corresponding to these different views are then combined mathematically, by a procedure which is both quantitative and free from arbitrary assumptions, to give the three dimensional structure in a tangible and permanent form. The method is most powerful for objects containing symmetrically arranged subunits, for here a single image effectively contains many different views of the structure. The symmetry of such an object can be introduced into the process of reconstruction, allowing the three dimensional structure to be reconstructed from a single view, or a small number of views. In principle, however, the method is applicable to any kind of structure, including individual unsymmetrical particles, or sections of biological specimens. The approach was now wholly computational. Computers were by this time fairly widely used in structural biology for computing electron density maps from X-ray crystallographic diffraction data, and it is no accident that three-dimensional reconstruction was invented in the laboratory that had earlier seen pioneering work in the development of protein crystallography. The optical density on the micrographs had first to be converted to a regularly sampled array of numbers, and this was achieved by a computer-controlled densitometer of the kind then being developed for measuring X-ray diffraction photographs (23). The core of the reconstruction method depended on a relationship well known to crystallographers, namely that the two-dimensional Fourier transform of a projection of a three-dimensional structure corresponds to the equivalent central section through the three-dimensional transform. Thus, different projected views of the object gave different central sections; with sufficient views the threedimensional transform could be filled in completely and the density in the object recovered by Fourier inversion (Fig. 7). The precise meaning of filled in completely depends both on the size of the object and on the degree of detail desired in the reconstructed map. The relationship is given by n?pd/d, where n is the number of equally spaced views needed to reconstruct an object of diameter D to a fineness of detail d. Although this formula was not quoted explicitly in the paper, it was used to generate a table of the number of views needed to reconstruct a helical object, an icosahedral virus and an object with no symmetry. Unlike the situation in protein crystallography, in which the X-ray diffraction pattern gives the strengths of the various Fourier components but not their relative phases, which have to be recovered indirectly by, for example, by adding heavy atoms to the protein, there is no phase problem in electron microscopy. The objective lens of the microscope recombines the diffracted beams from the specimen to create an image on the micrograph, in which the phase information representing the relative positions of the different spatial frequencies is recorded. This information is recovered numerically by computing the complex Fourier transform of the image, which then carries both amplitudes and phases of the Fourier components. DeRosier and Klug, having approached the problem from the perspective of optical filtering, did initially try to measure the phases optically by interference or holography. However, this proved very tedious and it became

7 VIRUSES AND DEVELOPMENT OF ELECTRON MICROSCOPY 245 Figure 7. The overall scheme for three-dimensional reconstruction (22), showing how images representing projections of the structure give sections of the three-dimensional Fourier transform. Once sufficient views have been included, the threedimensional map can be calculated by Fourier synthesis. Reprinted with permission from Nature Publishing Group. clear to them that the way to proceed was computationally, not optically. The greater power and flexibility of numerical image processing meant that henceforth optical methods were used only for a quick initial assessment of the quality of micrographs. The case of a helical structure was special, because the specimen effectively contained an inbuilt tilt axis, so that a single view of the tail presented a set of equally spaced views of the constituent subunit. This meant that the two-dimensional transform of a single image contained all the information required to make a three-dimensional map, at least to a limited degree of detail. This was expressed and extracted using the theory already developed for analysis of X-ray fibre diffraction patterns of helical specimens, and the three-dimensional map was actually computed by using the computer program written for making a map of TMV from X-ray diffraction data, again underlining the close interplay at that time between X-ray methods and electron microscopy. The map itself was visualized as a three-dimensional balsawood model, which is now housed in the Science Museum in London, as befits the first example of a completely new approach to biological structure determination (Fig. 8). For non-helical objects it was necessary to combine information from more than one image of the specimen. These images could either be of different particles lying at different orientations in the field of view or be collected from a single particle by tilting it in the microscope. In the latter case the amount of information that can be collected is limited by radiation damage to the specimen. In either case any internal symmetry in the particle is a great help in reducing the number of different views required and also in facilitating the computation. Accordingly, the next development and application of image reconstruction was to icosahedral viruses. With icosahedral symmetry, one view of a particle gives rise to 60 symmetry-related planes of data in the three-dimensional Fourier transform. However, in contrast with helical symmetry, the data are very unevenly distributed, a feature that is exacerbated by the inclusion of data from multiple views in arbitrary directions. Methods for combining and interpolating such unevenly sampled data were described by Crowther et al. (24) and applied to human wart virus and tomato bushy stunt virus (Fig. 9) (25). The latter paper also introduced the idea of common lines, lines of data that recur in the transform of a symmetrical particle. These can be used to find the orientation and centre of each particle, relative to its symmetry axes, which it is essential to know before the data can be combined. Cross common lines between particles can be used for interparticle scaling and ensuring that all the views are combined with a consistent handedness. The common lines residuals give a measure of how well preserved any given particle is. These ideas, although introduced in the special context of icosahedral viruses, have proved to have more general applicability in reconstruction procedures for particles of all kinds. THE USE OF UNSTAINED SPECIMENS The next major advance in electron microscope technique did not involve viruses but must be described here. The limitations of negative staining have already been referred to, in particular the fact that it does not reveal internal features of protein structure. Henderson and Unwin (26) introduced a completely new way of preparing specimens of two-dimensional crystals by embedding them on the grid in a coat of

8 246 CROWTHER Figure 8. Three-dimensional map of the T4 phage tail (22). The density in the map is represented by a set of sections consisting of glued balsawood cutouts. Prominent helical grooves on the surface, between the constituent protein subunits, and internal helical tunnels at an inner radius are clearly seen. Reprinted with permission from Nature Publishing Group. Figure 9. Map of tomato bushy stunt virus (25), in which the 180 protein subunits are clustered in 90 dimers. In (a) some of the dimeric units are indicated, and in (b) the positions of the icosahedral symmetry axes are marked. Some of the particles shown in Fig. 1e were used. Reprinted with permission from Nature Publishing Group. dried glucose. This preserves the specimen in a friendly environment and in a form suitable to be put into the microscope. However, it also generates a specimen that is of low contrast and very sensitive to radiation. The micrographs have to be taken at a very low electron dose and the images look featureless, being dominated by the fluctuations in the small number of electrons recorded in each image element. Nevertheless, if such a micrograph is put into an optical diffractometer, it is immediately apparent from the resulting array of spots that high-resolution information about the crystal has been recorded. This can be recovered by image averaging over the repeating unit cells of the crystal, using the computational equivalent of optical filtering. Provided the crystals are large enough, so that very large numbers of molecules are included in the averaging, very detailed information, out to roughly atomic spacings, can be recovered above the noise level. To collect three-dimensional information the grids must be tilted in the microscope to generate the different views needed. It is also necessary to take the pictures with a relatively high amount of defocus, to introduce phase contrast into the image. This is a rather imperfect way of producing contrast and detailed corrections have to be made, combining images taken with different levels of defocus, in the way described earlier by Erickson and Klug (27). Using this approach Henderson and Unwin (26) showed that the membrane protein bacteriorhodopsin, a light-driven proton pump, contained seven a-helical membrane-spanning regions. This was the first time that detailed information about the internal structure of a protein had been recovered by electron microscopy. The technique was pursued and improved by Henderson and colleagues, who eventually produced an atomic model of bacteriorhodopsin (28). But to return to viruses: the method of embedding the specimen in glucose was not really suitable for creating unstained specimens of individual particles, and a different approach was needed. This was established by Dubochet and his colleagues at the European Molecular Biology Laboratory in Heidelberg (29). What they did was to put the virus in buffer onto a holey carbon support film on the microscope grid, blot the drop to leave a thin layer of liquid and then rapidly plunge the grid into liquid ethane. The layer freezes so quickly that there is no time for ice crystals to form and the virus particles become embedded in solid glass-like water or vitreous ice. Inside the microscope one is therefore imaging particles suspended in a thin layer of vitreous ice over holes in the carbon film, so there is no interference from the support. To avoid crystallization of the water, the grid must be maintained at the temperature of liquid nitrogen, both during transfer into the microscope and during the collection of the micrographs. This technique has come to be known as electron cryomicroscopy of single particles, to distinguish it from work on crystalline specimens, which can also be prepared in this way. As with glucose embedding, the specimens are very sensitive to radiation and of low contrast, so they have to be imaged with low electron doses and relatively large levels of defocus. Among the earliest particulate specimens examined in this way by Dubochet and colleagues was a range of different viruses, including adenovirus, and spectacular images of the native structures were obtained (30). However, to extract detailed three-dimensional information from such images extensive computer processing is needed. A map of adenovirus at 35 A resolution was published in 1991 (31), showing a degree of detail far beyond the pingpong ball model of Fig. 1d.

9 VIRUSES AND DEVELOPMENT OF ELECTRON MICROSCOPY 247 Figure 10. Hepatitis B virus core protein shell (32). (a) Map computed from about 6400 particle images. The tubular features constituting the spikes on the surface are a-helices. (b) The fold of the protein subunit deduced from the map, with cylinders representing a-helical regions. The complete shell is made from 240 such subunits, paired to form 120 spikes. Reprinted with permission from Nature Publishing Group. Using this method of preparation and improved and modified versions of the computational methods originally developed for negatively stained icosahedral viruses, it proved possible to compute a map of the core protein shell of human hepatitis B virus (Fig. 10) to a sufficient degree of detail (7.4 Å resolution) to trace the path of the polypeptide chain in the subunit (32). This involved combining images of about 6400 particles from 34 micrographs at different levels of defocus, many more than the three or four images originally used for negatively stained specimens. Unusually for a virus capsid, the fold was largely a-helical, so it was possible to number the amino acids along the chain approximately. The fold and numbering were subsequently confirmed by X-ray crystallography (33). A virus sample thus provided the first example of a protein fold being solved by single-particle cryomicroscopy. CONCLUSION I hope I have given an impression of how, over the past 50 years, advances in techniques for specimen preparation and analysis of the resulting micrographs have transformed electron microscopy from a position where biological particles, such as viruses, could just barely be visualized to one where significant details of internal structure can be established. Viruses have provided the test specimen of choice for the majority of these advances, although the resulting methods are now being productively applied to specimens of all kinds. The developments described have been underpinned by parallel improvements in the microscopes themselves and their associated instrumentation, as well as in computer programs and algorithms, film scanners and displays. What of the future? As Henderson has argued (34), there is no intrinsic reason why electron microscopy should not be able to produce details of single particles at atomic resolution, as has already been achieved for two-dimensional crystals. All that is currently limiting the micrographs is too fast a fall off in signal : noise ratio with increasing resolution. If this can be improved, and provided that the particles are large enough for their individual orientations and positions to be determined accurately, then atomic imaging of particulate specimens could become a reality. Will a virus sample again provide the critical test specimen? ACKNOWLEDGEMENTS The idea for this paper came as a result of a lecture delivered when I was a Burroughs Wellcome Fund Visiting Professor at Baylor College of Medicine in Houston, and I thank Professor Wah Chiu and his colleagues for their hospitality. I am grateful to Dr Finch, Dr Henderson and Sir Aaron Klug for their helpful comments on the manuscript, and to Annette Lenton for help in preparing the figures. REFERENCES 1. Ruska, E. The development of the electron microscope and of electron microscopy [Nobel lecture]. Available at /ruska-lecture.pdf. 2. Ford, B. J. (2001) The Royal Society and the microscope. Notes Rec. R. Soc. Lond. 55, Hall, C. E. (1955) Electron densitometry of stained virus particles. J. Biophys. Biochem. Cytol. 1, Huxley, H. E. (1956) Some observations on the structure of tobacco mosaic virus. First Eur. Conf. on Electron Microscopy, Stockholm, pp Brenner, S., and Horne, R. W. (1959) A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, Brenner, S. (1997) A life in science. p. 118, Current Science Group, London. 7. Caspar, D. L. D. (1966) An analogue for negative staining. J. Mol. Biol. 15, Horne, R. W., Brenner, S., Waterson, A. P., and Wildy, P. (1959) The icosahedral form of an adenovirus. J. Mol. Biol. 1, Crick, F. H. C., and Watson, J. D. (1956) Structure of small viruses. Nature 177, Caspar, D. L. D., and Klug, A. (1962) Physical principles in the construction of regular viruses. Cold Spring Harb. Symp. Quant. Biol. 27, Klug, A., and Finch, J. T. (1965) Structure of viruses of the papilloma polyoma type. I. Human wart virus. J. Mol. Biol. 11, Klug, A., and Finch, J. T. (1968) Structure of viruses of the papilloma polyoma type. IV. Analysis of tilting experiments in the electron microscope. J. Mol. Biol. 31, Markham, R., Frey, S., and Hills, G. J. (1963) Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20, Galton, F. (1878) Composite portraits. Nature 18,

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