260
Biochimica et BiophysicaActa, 1038 (1990) 260-267
Elsevier BBAPRO 33622
Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing George Harauz and Derrick Flannigan Department of Molecular Biology and Genetics, Universityof Guelph, Guelph, Ontario (Canada)
(Received 18 August 1989)
Key words: Ribosome;Thermophile;Fungi; Electronmicroscopy;Imageprocessing; (T. lanuginosus)
Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution. The overlap and non-overiap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition. The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species. Additionally, the top view has not been previously described for eubacteria. No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms.
Introduction Ribosomes from the eubacterium Escherichia coli have been investigated extensively and form the basis for our picture of ribosome structure and function [1,2]. This view of the ribosome has been obtained by electron microscopy and other biophysical techniques (e.g., neutron scattering). Although, transmission electron microscopy provides images of macromolecular complexes directly, the inherent noisiness of electron micrographs limits the detail that one can interpret visually. The appearance of ribosomes in electron images of negatively stained preparations is influenced by a number of factors: (i) the three-dimensional structure; (ii) the ionic composition of the buffers employed in the preparations; (iii) biological microheterogeneity; and (iv) the protein and RNA composition, which may result in some positive staining. Although susceptible to artefact and misinterpretation, electron microscopy nonetheless remains a valuable tool for probing ribosome structure, especially when coupled to computerised image processing techniques. In particular, multivariate statistical analysis and classification algorithms facilitate the sorting of images according to their principal features [3-5]. Images of different com-
Correspondence: G. Harauz, Department of Molecular Biologyand Genetics, Universityof Guelph, Guelph, Ontario, N1G 2W1, Canada.
plexes that lie in a similar orientation upon the specimen support film can be averaged to give characteristic views with an enhanced signal-to-noise ratio and better spatial resolution [6]. Ribosomal subunits from different bacterial species have recently been compared by electron microscopy and image analysis [7,8] as a first step toward comparing the actual three-dimensional structures. The initial application of the sorting algorithms to electron images of the small ribosomal subunits from E. coli and the thermophilic eubacterium Bacillus stearothermophilus yielded constructions of three predominant views of the complex for both species, to a reproducible spatial resolution of 1.7 nm [7]. More recently, the characteristic views of the large subunits from E. coli and the archaebacteria Methanococcus vannielii, Sulfolobus solfataricus and Halobacterium marismortui were studied [8] to a resolution approaching 2.0 nm. Although the gross structural morphology was similar, significant differences in the features between subunits from the different prokaryotes were discernible. Eukaryotic ribosomes have received relatively little attention compared with those from prokaryotes, and to date no comparative study employing image processing has been performed. In this paper, we describe the computational analysis of electron micrographs of ribosomes from the eukaryotic thermophile Thermomyces lanuginosus [9-11], a fungus which grows optimally at a temperature of about 55 ° C. The enhanced
0167-4838/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
261 thermostability of ribosomes in this organism may be reflected in their morphology [7,12,13]. The results demonstrate, however, that the ribosomes from this thermophilic fungus have a three-dimensional architecture closely related to that observed for ribosomes from the mesophilic eubacterium E. coli, indicating that the major structural features of these macromolecular complexes are strongly conserved across significant phylogenetic gaps. Materials and Methods
Isolation and characterisation of ribosomes Th. lanuginosus was grown in starch broth at 55 ° C with agitation for 60-72 h and the mycelium was harvested by filtration. All subsequent steps were carried out at 4 o C, using autoclaved buffers and glassware. The mycelium (2.5-3 g) was ground with alumina powder (5 g) for 10-15 min using a mortar and pestle, after which 4 volumes of homogenisation buffer (20 m M Tris-HC1 (pH 7.5), 5 mM Mg (CH3COO)2, 10 mM KC1, 6 mM 2-mercaptoethanol, 25 units/ml RNAguard (Pharmacia)) were added and mixed. The sample was centrifuged at 8000 rpm (5000 x g) in a Beckman JA20 rotor for 30 min. The supernatant was then removed and centrifuged again at 18000 rpm (25 300 x g) for 45 min. This supernatant was layered over a cushion of 30% sucrose in homogenisation buffer, in a volume ratio of 2 : 1, and centrifuged at 35 000 rpm (93 000 × g) for 20 h in a Beckman 70 Ti rotor. The pellet, which contained ribosomes, was rinsed three times with sterile double-distilled water, and resuspended in 1 ml of homogenisation buffer. The nucleic acid composition of the ribosome fraction was assayed by electrophoresis on 3% polyacrylamide and 1.2% agarose gels.
Electron microscopy Isolated ribosomes were prepared for electron microscopy on a single layer carbon film by the adsorption technique [4] and negatively contrasted with 2% uranyl acetate. Electron micrographs were taken on a JEOL 100CX at an instrument magnification of 33 000 x and an accelerating voltage of 60 kV (Fig. 1). The grids were consistently inserted into the microscope with the specimen side oriented away from the electron source. Each specimen area was not preilluminated prior to being micrographed, to minimise the total electron dose.
Fig. 1. Electron micrograph of Th. lanuginosusribosomes, negatively contrasted with uranyl acetate. Scalebar = 100 nm.
processing system [14]. All distinct complexes that were not in close contact with others were selected interactively using the IRIS monitor and mouse controlled cursor. A total of 815 particles was selected, i.e., 815 subimages of size 64 x 64 pixels and containing a single ribosome within them were extracted from the larger micrographs. Subsequent data analysis steps have been previously described in detail [4,8] and are summarised here. The single particle images were pretreated by band-pass filtering, surrounded by a circular mask, floated within this mask to a zero average density and given a normalised variance. Certain images were selected after an exhaustive visual survey to use as references for alignment (Fig. 2). The entire data set was aligned with respect to each of these references in turn. The final
I
Image analysis Five electron micrographs were digitised using an Optronics rotating drum densitometer and a scanning step size of 25 #m, corresponding to 0.76 nm at the object level. Digitised images were transferred via magnetic tape to an IRIS 3120 workstation (Silicon Graphics, Mountain View, CA). Data analysis was performed in the framework of the I M A G I C image
Fig. 2. Images of individual ribosomes that were selected from the total data set of 815 particles and used as references (after centering and contouring) for the initial multi-referencealignment step.
262 alignment parameters for each image were then chosen on the basis of highest correlation coefficient between the aligned and reference images. Then multivariate statistical analysis and classification were performed [4,5]. A mask was formed from the total sum of the aligned images to limit the analysis to only those areas containing relevant structural information. The multivariate statistical analysis decomposed the set of images, into its principal components, or eigenimages. The first 24 of these were calculated, as is the routine in IMAGIC. The lower order components described most of the interimage variation and were used to group together those images that most resemble each other using a hierarchical ascendant classification algorithm [5]. Twelve eigenimages with weighting defined by the inverse square root of the corresponding eigenvalue were used in the classification, and 15% of the input images were rejected as representing either misaligned complexes or uncharacteristic views. The data set was partitioned into 30 classes; the number of classes was chosen so that each class would have about 20 members on average. The images comprising each class were summed together to give a 'class average' in which sources of random noise (e.g., stain variations) were averaged out to become less significant compared to the common signal, viz., the projected structure of the ribosome in a specific orientation. In the next stage of the analysis, four class averages were selected and used as references, along with their mirror views, in a new set of multireference alignments
Fig. 3. Averagesof ribosomeimages that were derived from the initial alignment, multivariate statistical analysis and classification steps, and used as referencesfor the second multi-referencealignment. The number of members comprisingeach class is (a) 51, (b) 36, (c) 27 and (d) 40. Images that aligned well with reference (c) resulted in an overlap class that was rotated by 180°, a result corrected in the final presentation (Fig. 4).
(Fig. 3). The newly aligned data set then underwent multivariate statistical analysis and classification again. The class averages obtained after this refinement step are the ones reported here. Results and Discussion
Isolation and electron microscopy Electron microscopy of ribosomes from Th. lanuginosus (Fig. 1) indicated a homogeneous population of particles. Gel electrophoresis of extracted rRNAs (data not shown) revealed the presence of fragments distinct from the 23 and 16 S E. coli rRNA markers, and presumably corresponding to the expected 25, 18, 5.8 and 5 S eukaryotic rRNAs [10,11]. The homogeneity of the sample in the electron micrographs implied that little degradation had occurred during the isolation procedure, which was designed to minimise endogenous enzymatic activity. The Th. lanuginosus ribosomes (Fig. 1) are approx. 24.5-26.5 nm in diameter, marginally bigger than those from E. coli (about 23 nm). They lack distinctive structural features, showing pentagonal or slightly prolate contours and varying regions of internal negative staining. After digitisation and particle extraction, the data set was scanned visually and six images were selected as references for the initial alignment step (Fig. 2). The small and large subunits shown in Fig. 2 appear distinct in three cases, while their contours cannot be discerned in the other three. In Figs. 2-5, it should be noted that intermediate spatial frequency detail has been emphasised by the initial band-pass filtering operation. Sorting of characteristic views After the initial alignment, a multivariate statistical analysis and classification of the data set was performed and 30 class averages constructed. Two primary criteria were used to rank these: (i) the variance of pixels within each mask, and (ii) the number of members within each class. The four 'best' and distinct class averages were then used along with their mirror images in a new set of multi-reference alignments. Again, a multivariate statistical analysis and classification decomposed the population into 30 classes. Of these, 21 were rejected as having too few members or too large an intraclass variance. The rejected classes included a number that appeared to represent mirror views of the references; however, the class members were found to be heterogeneous even on visual examination and represented mainly poorly aligned images. The class averages that were considered to be the best by these relatively stringent criteria are shown in Fig. 4. These represent the predominant projection forms of Th. lanuginosus ribosomes, and now can" easily be grouped together on the basis of obviously similar fea-
263
Fig. 4. Selectedclass averagesderived from the final MSA and classification,grouped according to view: overlap (1-5), non-overlap(6 and 7) and top (8 and 9). Images are in order of increasing intraclass variance within each grouping. The number of images comprisingclasses 1-9 is 46, 29, 31, 34, 22, 35, 32, 27 and 27, and the values of intraclass variance are (in arbitrary units) 60.0, 60.1, 62.5, 64.3, 68.5, 65.1, 67.7, 63.5 and 70.1, respectively.
tures. There are three main views, henceforth referred to as overlap, non-overlap and top, indicating the perceived relationship between the constituent subunits. The overlap and non-overlap views are similar in composition to two defined views of the E. coli ribosome [15], and thus it is reasonable to retain the terminology. The top view has not previously been described in the literature. The most commonly occurring view in our population of 815 individual particles is the non-overlap view: classes 1-5 in Fig. 4, consisting of 46, 29, 31, 34 and 22 members, respectively. Although there were two other non-overlap classes determined, they were of high intraclass variance and contained only 16 and 17 members (data not shown). The small subunit is clearly recognisable lying to the right in lateral projection. Fig. 5 shows the constituent images comprising class 1. Although they appear similar to the eye, their sum shows structural detail far better.
Another commonly occurring view is the overlap view: classes 6 and 7 in Fig. 4, consisting of 35 and 32 members, respectively. Three other overlap classes were also determined in the analysis, but are not shown here since they consisted of only 17, 14 and 15 members. In studies of E. coli ribosomes [15-19], the stain excluding body on the left has been shown to represent the small subunit, with the large subunlt lying to the right and below. The least frequently occurring view is what we call the top view: classes 8 and 9 in Fig. 4, each consisting of 27 members. Another top class of 22 members, but with a high intraclass variance, is not shown. As in the non-overlap view, the profiles of the two subunits are distinct. In the top projection, we interpret the particle on the right to be the large subunit because it is the larger stain-excluding region and has a rounded hemispherical appearance. The particle on the left has a characteristic head and collar (in class 9) and a cylin-
264
Fig. 5. The 46 individualimagescomprisingoverlap class 1. Their averageis image 1 in Fig. 4.
drical profile, suggesting that it is the small subunit. Reflecting the top view through a vertical axis brings the two subunits into the same relative positions as in the non-overlap view. This mirrored top view then appears to differ from the non-overlap projection by a rotation about an axis passing through the two subunits and in the plane of the image. Unfortunately, we were unable to prove these conjectures using angular reconstitution techniques [20]. The total numbers of complexes lying in the nonoverlap, overlap and top orientations is 195 (24% of 815), 113 (14%) and 76 (9%), respectively. The relative proportions of non-overlap/overlap/top views is thus 51:29:20. The non-overlap projection has the lowest intraclass variance (Fig. 4), indicating it is a stable position in which the ribosome can adsorb to the carbon. The overlap and top projections have generally higher intraclass variances, perhaps indicating that they may 'rock' about a particular orientation.
Spatial resolution of characteristic views Visually, the quality of the class averages is much better than of the original images. An objective measure
of the resolution of detail in these images is obtained by the Fourier Ring Correlation method [6]. This method assesses the similarity between independent images by determining the cross-resolution, i.e., the value of the highest common spatial frequency present in the two images. Clearly, the comparison must be between similar image, either (a) independent class averages of the particle in the same orientation, (b) two 'half-averages' obtained by summing odd and even numbered members of a particular class or (c) any two members of a particular class. The cross-resolution between any two images comprising class 1 (Fig. 5) is approx. 7.5 nm. The two averages were formed by adding alternate class members, and the cross-resolution between the two sums was 1.8 nm, indicating the significant improvement attained by averaging. This figure is best interpreted as a resolution limit attained in this particular class. The reproducible spatial resolution between any two pairs of non-overlap views in Fig. 4 averages 3.7 nm, and ranges from 1.8-5.7 nm (there are 10 combinations of pairs). For both the overlap and top views, the calculated value for the reproducible spatial resolution is 3.9 nm.
265
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respectively. The non-overlap class occurs as a cluster, distinct from the rest of the population, at the top left of the plot. The overlap views form a relatively compact group at the top right, while the individual top views are more sparsely distributed in the central region of this space. The major group of images at the bottom of the plot in Fig. 6b consisted primarily of misaligned images; although these were clustered by the classification algorithm their classes were subsequently rejected from further consideration. The plots in Fig. 6 are defined only by two factorial coordinates, whereas 12 coordinates were used in image sorting. The compactness or sparseness of classes seen in the two-dimensional space should be interpreted with caution•
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Fig. 6. (a) Eigenimages 1, 2 and 3, representing the basis vectors corresponding to factorial coordinates 1, 2 and 3. These eigenimages account for 17.1, 6.3 and 3.3% of the total interimagevariancein the data set. (b) Plot of positions of 815 ribosomeimagesin a two-dimensional space representedby factorialcoordinates2 and 3. Each point represents 1 image•Plots of positions of imagescomprising(c) class 1 (non-overlap), (d) class 6 (overlap) and (e) class 8 (top). Meaning of eigenimages The first 12 eigenimages calculated during the multivariate statistical analysis accounted for 42.6% of the total interimage variance, while all 24 accounted for 53.7%. The hierarchical ascendant classification did not give significantly different results if 12 or 24 factorial coordinates were used for the clustering; the results presented here were obtained by grouping in a 12-dimensional space. The first eigenimage (Fig. 6a) points to the center of mass of the entire data set, and resembles the predominant (non-overlap) projection. The second eigenimage appears to define the difference between the non-overlap and other views, especially with respect to the stain-excluding region occupied by the small subunit, and the off-center stain pit. The third eigenimage characterises less easily described differences in ribosome size, shape and internal stain distribution. A plot of the positions of all 815 images in a two-dimensional space, defined by factorial coordinates 2 and 3, is shown in Fig. 6b. Three major groupings are suggested: at the top left, the top right, and the bottom of the plot. Figs. 6c-e show only the images comprising class 1 (non-overlap), class 6 (overlap) and class 8 (top),
Comparison with E. coli 70 S monosome structure The 70 S ribosome of E. coli was surveyed in the electron microscope by Lake [15]. Two main views of the particle were distinguished, called overlap or nonoverlap, corresponding to whether the profiles of the large and small subunits overlapped. Boublik et al. [16] investigated technical factors of specimen preparation, but analysed only one particular view corresponding to Lake's overlap view. The relative orientation of the two subunits was examined by immunoelectron microscopy [17,18]• More recently, Verschoor et al. [19] applied image sorting and averaging algorithms to electron images of E. coli 70 S ribosomes. It is worth describing their analysis technique in detail, since it differs significantly in many respects from our own work here. Verschoor and colleagues studied electron micrographs of 70 S particles from E. coli using a double-carbon 'sandwich' preparation [2,16]. They selected a total of 308 individual particles, classified these visually into L, O and R types (73, 89 and 146 particles, respectively), and formed averages of the subsets. The L type is the non-overlap view, while the O and R types correspond roughly to Lake's original overlap view. Using a combination of principal components analysis (with only eight factorial coordinates) and electron micrographs of tilted specimens, it was shown that the O and R types related to endpoints of a continuum of rotation-related views spanning about 50 degrees. These results were later confirmed by Frank et al. [21] using a hybrid clustering algorithm. In this paper, we describe an analysis of electron micrographs of Th. lanuginosus ribosomes, negatively stained and lying on a single carbon layer• The ribosomes from this organism appear similar in outline and internal staining pattern to those from the eubacterium E. coli, although the rRNA compositions are different. Th. lanuginosus ribosomes adsorb to the grid in one of three preferred ways, giving three distinct projections; two of these correspond to previously studied adsorption modes for E. coli ribosomes. The relative propor-
266 tions of complexes seen in different views are significantly different for the two species; the non-overlap form occurs most frequently for the thermophilic fungus, whereas the overlap form is commonest for the eubacterium. It was not possible to distinguish subsets of overlap views of Th. lanuginosus ribosomes differing by a single axis rotation, because of the relatively small number of particles lying in this position, as well as the limited spatial resolution of the images used in this study. The Th. lanuginosus ribosomes also lie on the carbon film in another orientation, the top view, which appears to be related to the non-overlap view by a rotation about an axis perpendicular to the long axes of the constituent subunits. This projection form occurs infrequently, although it has been observed in analyses of large data sets of E. coli ribosomes (Boekema, E., Harauz, G. and Van Heel, M., unpublished data). Perhaps the key factors to its elucidation were: (i) the use of a single carbon preparation [4,16] and (ii) the relatively large size of the data set subjected to multivariate statistical analysis.
Comparison with other eukaryotes and thermophiles Eukaryotic ribosomes have not been studied by electron microscopy to as great an extent as those from prokaryotes, with emphasis having been on subunit structure [22,23]. Ribosomes from the vegetative amoebae and spores of the slime mold Dictyostelium discoideum have been imaged, but sophisticated image processing techniques were not available at that time [24]. The D. discoideum ribosomes were about 26 nm in size and clear non-overlap views were distinguished. Other studies of three-dimensional reconstructions of ribosomes from chick embryos to 5.5 nm resolution enabled the localisation of the exit channel for nascent protein, but these images cannot be directly compared with those of the Th. lanuginosus ribosomes derived here [25]. The only statement that can be made at present is that the mutual orientations of the large and small subunits in eubacterial and eukaryotic ribosomes are the same. The structures of ribosomal subunits from one other thermophilic organism, the eubacterium Bacillus stearothermophilus, have been studied [7,13]. This organism grows optimally at 65°C. Although subtle differences when compared to E. coli ribosomes were detected, it is not clear how these morphological alterations are related to thermophily. It is perhaps not surprising that ribosomes from a moderate thermophile may have even less easily detectable structural modifications than those from an extreme one. In general, though, macromolecular complexes isolated from thermophiles are better suited for structural studies, forming crystals in a time span in which their mesophilic counterparts are degraded [13,26]. Moreover, since con-
formational differences in functionally or compositionally distinct ribosomes may be less than 1 nm in size (e.g., Ref. 24 and 27), a higher resolution study is required. This may involve processing of electron images of unstained specimens, or attempts at crystallisation and X-ray diffraction studies. Our immediate future investigations on Th. lanuginosus ribosomes will include separation and analysis of the individual subunits to higher resolution by imaging at a higher magnification, and three-dimensional reconstruction from the characteristic projection views using micrographs of tilted specimens [3,28]. This approach will help to determine accurately the relative orientations between the different classes.
Conclusions Ribosomes from the thermophile Th. lanuginosus have been examined by electron microscopy and image analysis of negatively stained preparations. The complexes were about 24.5-26.5 nm in size, and 3 predominant projection forms were constructed to a reproducible spatial resolution approaching 1.8 nm. The characteristic projections of Th. lanuginosus ribosomes are similar in gross appearance to those described in the literature from the eubacterium E. coli, but occur in different proportions. This reflects the complexes' protein and rRNA compositions, which affect their adsorption behaviour. Despite the evolutionary distance between these two organisms, their differing protein and rRNA content, and the proven ability of our algorithmic approach to demonstrating subtle differences in structural features of ribosomes [4], quaternary ribosome structure is highly conserved, indicating its important relation to ribosome function.
Acknowledgements This work was supported by grants from the Research Advisory Board of the University of Guelph, and the Natural Sciences and Engineering Research Council of Canada to G.H. We are grateful to Dr. Patrick Whippey (University of Western Ontario) for assistance with densitometry, to Dr. Dawn Larson and Dr. Peter Zahradka for their comments on the manuscript, and to a reviewer for comments on the interpretation of the top view.
References 1 Wittmann, H.-G. (1983) Annu. Rev. Biochem. 52, 35-65. 2 Stoeffler-Meilicke, M. and Stoeffler, G. (1988) Methods Enzymol. 164, 503-520. 3 Frank, J., Radermacher, M., Wagenknecht, T. and Verschoor, A. (1988) Methods Enzymol. 164, 3-35. 4 Harauz, G., Boekema, E.J. and Van Heel, M.G. (1988) Methods Enzymol. 164, 35-49.
267 5 van Heel, M. (1984) Ultramicroscopy 13, 165-183. 6 Van Heel, M. (1987) Ultramicroscopy 21, 95-100. 7 Van Heel, M. and Stoeffler-Meilicke, M. (1985) EMBO J. 4, 2389-2395. 8 Harauz, G., Stoeffler-Meilicke, M. and Van Heel, M. (1987) J. Mol. Evol. 26, 347-357. 9 Domsch, K.H., Gains, W. and Anderson, T.-H. (1980) Compendium of Soil Fungi, Academic Press, New York. 10 Nazar, R.N. and Wildeman, A.G. (1983) Nucleic Acids Res. 11, 3155-3168. 11 Nazar, R.N., Wong, W.M. and Abrahamson, J.L.A. (1987) J. Biol. Chem. 262, 7523-7527. 12 Miller, H.M. and Shepherd, M.G. (1973) Can. J. Microbiol. 19, 761-763. 13 Yonath, A. and Wittmann, H.-G. (1988) Methods Enzymol. 164, 95-107. 14 Van Heel, M. and Keegstra, W. (1981) Ultramicroscopy 7, 113130. 15 Lake, J.A. (1976) J. Mol. Biol. 105, 131-159. 16 Boublik, M., Hellmann, W. and Kleinschmidt, A.K. (1977) Cytobiologie 14, 293-300.
17 Kastner, B., Stoeffler-Meilicke, M. and Stoeffler, G. (1981) Proc. Natl. Acad. Sci. USA 78, 6652-6656. 18 Lake, J.A. (1982) J. Mol. Biol. 161, 89-106. 19 Verschoor, A., Frank, J., Wagenknecht, T. and Boublik, M. (1986) J. Mol. Biol. 187, 581-590. 20 Van Heel, M. (1987) Ultramicroscopy 21, 111-124. 21 Frank, J., Bretaudiere, J.-P., Carazo, J.-M., Verschoor, A. and Wagenknecht, T. (1988) J. Microsc. 150, 99-115. 22 Frank, J., Verschoor, A. and Boublik, M. (1981) Science 214, 1353-1355. 23 Lutsch, G. and Bielka, H. (1988) Eur. J. Biochem. 172, 653-662. 24 Boublik, M. and Ramagopal, S. (1980) Mol. Gen. Genet. 179, 483-488. 25 Milligan, R.A. and Unwin, P.N.T. (1986) Nature 319, 693-695. 26 Morikawa, K., Fujiyoshi, Y., Ishizuka, K., Sugiyama, J., Kawakami, M. and Takemura, S. (1986) J. Microsc. 142, 247-258. 27 Vasiliev, V.D., Selivanova, O.M., Baranov, V.I. and Spririn, A.S. (1983) FEBS Lett. 155, 167-172. 28 Wagenknecht, T., Radermacher, M. and Frank, J. (1987) in Fifth Conversation in Biomolecular Stereodynamics, pp. 251-252, Adenine Press, New York.
Biochimica et BiophysicaActa, 1038 (1990) 260-267
Elsevier BBAPRO 33622
Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing George Harauz and Derrick Flannigan Department of Molecular Biology and Genetics, Universityof Guelph, Guelph, Ontario (Canada)
(Received 18 August 1989)
Key words: Ribosome;Thermophile;Fungi; Electronmicroscopy;Imageprocessing; (T. lanuginosus)
Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution. The overlap and non-overiap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition. The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species. Additionally, the top view has not been previously described for eubacteria. No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms.
Introduction Ribosomes from the eubacterium Escherichia coli have been investigated extensively and form the basis for our picture of ribosome structure and function [1,2]. This view of the ribosome has been obtained by electron microscopy and other biophysical techniques (e.g., neutron scattering). Although, transmission electron microscopy provides images of macromolecular complexes directly, the inherent noisiness of electron micrographs limits the detail that one can interpret visually. The appearance of ribosomes in electron images of negatively stained preparations is influenced by a number of factors: (i) the three-dimensional structure; (ii) the ionic composition of the buffers employed in the preparations; (iii) biological microheterogeneity; and (iv) the protein and RNA composition, which may result in some positive staining. Although susceptible to artefact and misinterpretation, electron microscopy nonetheless remains a valuable tool for probing ribosome structure, especially when coupled to computerised image processing techniques. In particular, multivariate statistical analysis and classification algorithms facilitate the sorting of images according to their principal features [3-5]. Images of different com-
Correspondence: G. Harauz, Department of Molecular Biologyand Genetics, Universityof Guelph, Guelph, Ontario, N1G 2W1, Canada.
plexes that lie in a similar orientation upon the specimen support film can be averaged to give characteristic views with an enhanced signal-to-noise ratio and better spatial resolution [6]. Ribosomal subunits from different bacterial species have recently been compared by electron microscopy and image analysis [7,8] as a first step toward comparing the actual three-dimensional structures. The initial application of the sorting algorithms to electron images of the small ribosomal subunits from E. coli and the thermophilic eubacterium Bacillus stearothermophilus yielded constructions of three predominant views of the complex for both species, to a reproducible spatial resolution of 1.7 nm [7]. More recently, the characteristic views of the large subunits from E. coli and the archaebacteria Methanococcus vannielii, Sulfolobus solfataricus and Halobacterium marismortui were studied [8] to a resolution approaching 2.0 nm. Although the gross structural morphology was similar, significant differences in the features between subunits from the different prokaryotes were discernible. Eukaryotic ribosomes have received relatively little attention compared with those from prokaryotes, and to date no comparative study employing image processing has been performed. In this paper, we describe the computational analysis of electron micrographs of ribosomes from the eukaryotic thermophile Thermomyces lanuginosus [9-11], a fungus which grows optimally at a temperature of about 55 ° C. The enhanced
0167-4838/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
261 thermostability of ribosomes in this organism may be reflected in their morphology [7,12,13]. The results demonstrate, however, that the ribosomes from this thermophilic fungus have a three-dimensional architecture closely related to that observed for ribosomes from the mesophilic eubacterium E. coli, indicating that the major structural features of these macromolecular complexes are strongly conserved across significant phylogenetic gaps. Materials and Methods
Isolation and characterisation of ribosomes Th. lanuginosus was grown in starch broth at 55 ° C with agitation for 60-72 h and the mycelium was harvested by filtration. All subsequent steps were carried out at 4 o C, using autoclaved buffers and glassware. The mycelium (2.5-3 g) was ground with alumina powder (5 g) for 10-15 min using a mortar and pestle, after which 4 volumes of homogenisation buffer (20 m M Tris-HC1 (pH 7.5), 5 mM Mg (CH3COO)2, 10 mM KC1, 6 mM 2-mercaptoethanol, 25 units/ml RNAguard (Pharmacia)) were added and mixed. The sample was centrifuged at 8000 rpm (5000 x g) in a Beckman JA20 rotor for 30 min. The supernatant was then removed and centrifuged again at 18000 rpm (25 300 x g) for 45 min. This supernatant was layered over a cushion of 30% sucrose in homogenisation buffer, in a volume ratio of 2 : 1, and centrifuged at 35 000 rpm (93 000 × g) for 20 h in a Beckman 70 Ti rotor. The pellet, which contained ribosomes, was rinsed three times with sterile double-distilled water, and resuspended in 1 ml of homogenisation buffer. The nucleic acid composition of the ribosome fraction was assayed by electrophoresis on 3% polyacrylamide and 1.2% agarose gels.
Electron microscopy Isolated ribosomes were prepared for electron microscopy on a single layer carbon film by the adsorption technique [4] and negatively contrasted with 2% uranyl acetate. Electron micrographs were taken on a JEOL 100CX at an instrument magnification of 33 000 x and an accelerating voltage of 60 kV (Fig. 1). The grids were consistently inserted into the microscope with the specimen side oriented away from the electron source. Each specimen area was not preilluminated prior to being micrographed, to minimise the total electron dose.
Fig. 1. Electron micrograph of Th. lanuginosusribosomes, negatively contrasted with uranyl acetate. Scalebar = 100 nm.
processing system [14]. All distinct complexes that were not in close contact with others were selected interactively using the IRIS monitor and mouse controlled cursor. A total of 815 particles was selected, i.e., 815 subimages of size 64 x 64 pixels and containing a single ribosome within them were extracted from the larger micrographs. Subsequent data analysis steps have been previously described in detail [4,8] and are summarised here. The single particle images were pretreated by band-pass filtering, surrounded by a circular mask, floated within this mask to a zero average density and given a normalised variance. Certain images were selected after an exhaustive visual survey to use as references for alignment (Fig. 2). The entire data set was aligned with respect to each of these references in turn. The final
I
Image analysis Five electron micrographs were digitised using an Optronics rotating drum densitometer and a scanning step size of 25 #m, corresponding to 0.76 nm at the object level. Digitised images were transferred via magnetic tape to an IRIS 3120 workstation (Silicon Graphics, Mountain View, CA). Data analysis was performed in the framework of the I M A G I C image
Fig. 2. Images of individual ribosomes that were selected from the total data set of 815 particles and used as references (after centering and contouring) for the initial multi-referencealignment step.
262 alignment parameters for each image were then chosen on the basis of highest correlation coefficient between the aligned and reference images. Then multivariate statistical analysis and classification were performed [4,5]. A mask was formed from the total sum of the aligned images to limit the analysis to only those areas containing relevant structural information. The multivariate statistical analysis decomposed the set of images, into its principal components, or eigenimages. The first 24 of these were calculated, as is the routine in IMAGIC. The lower order components described most of the interimage variation and were used to group together those images that most resemble each other using a hierarchical ascendant classification algorithm [5]. Twelve eigenimages with weighting defined by the inverse square root of the corresponding eigenvalue were used in the classification, and 15% of the input images were rejected as representing either misaligned complexes or uncharacteristic views. The data set was partitioned into 30 classes; the number of classes was chosen so that each class would have about 20 members on average. The images comprising each class were summed together to give a 'class average' in which sources of random noise (e.g., stain variations) were averaged out to become less significant compared to the common signal, viz., the projected structure of the ribosome in a specific orientation. In the next stage of the analysis, four class averages were selected and used as references, along with their mirror views, in a new set of multireference alignments
Fig. 3. Averagesof ribosomeimages that were derived from the initial alignment, multivariate statistical analysis and classification steps, and used as referencesfor the second multi-referencealignment. The number of members comprisingeach class is (a) 51, (b) 36, (c) 27 and (d) 40. Images that aligned well with reference (c) resulted in an overlap class that was rotated by 180°, a result corrected in the final presentation (Fig. 4).
(Fig. 3). The newly aligned data set then underwent multivariate statistical analysis and classification again. The class averages obtained after this refinement step are the ones reported here. Results and Discussion
Isolation and electron microscopy Electron microscopy of ribosomes from Th. lanuginosus (Fig. 1) indicated a homogeneous population of particles. Gel electrophoresis of extracted rRNAs (data not shown) revealed the presence of fragments distinct from the 23 and 16 S E. coli rRNA markers, and presumably corresponding to the expected 25, 18, 5.8 and 5 S eukaryotic rRNAs [10,11]. The homogeneity of the sample in the electron micrographs implied that little degradation had occurred during the isolation procedure, which was designed to minimise endogenous enzymatic activity. The Th. lanuginosus ribosomes (Fig. 1) are approx. 24.5-26.5 nm in diameter, marginally bigger than those from E. coli (about 23 nm). They lack distinctive structural features, showing pentagonal or slightly prolate contours and varying regions of internal negative staining. After digitisation and particle extraction, the data set was scanned visually and six images were selected as references for the initial alignment step (Fig. 2). The small and large subunits shown in Fig. 2 appear distinct in three cases, while their contours cannot be discerned in the other three. In Figs. 2-5, it should be noted that intermediate spatial frequency detail has been emphasised by the initial band-pass filtering operation. Sorting of characteristic views After the initial alignment, a multivariate statistical analysis and classification of the data set was performed and 30 class averages constructed. Two primary criteria were used to rank these: (i) the variance of pixels within each mask, and (ii) the number of members within each class. The four 'best' and distinct class averages were then used along with their mirror images in a new set of multi-reference alignments. Again, a multivariate statistical analysis and classification decomposed the population into 30 classes. Of these, 21 were rejected as having too few members or too large an intraclass variance. The rejected classes included a number that appeared to represent mirror views of the references; however, the class members were found to be heterogeneous even on visual examination and represented mainly poorly aligned images. The class averages that were considered to be the best by these relatively stringent criteria are shown in Fig. 4. These represent the predominant projection forms of Th. lanuginosus ribosomes, and now can" easily be grouped together on the basis of obviously similar fea-
263
Fig. 4. Selectedclass averagesderived from the final MSA and classification,grouped according to view: overlap (1-5), non-overlap(6 and 7) and top (8 and 9). Images are in order of increasing intraclass variance within each grouping. The number of images comprisingclasses 1-9 is 46, 29, 31, 34, 22, 35, 32, 27 and 27, and the values of intraclass variance are (in arbitrary units) 60.0, 60.1, 62.5, 64.3, 68.5, 65.1, 67.7, 63.5 and 70.1, respectively.
tures. There are three main views, henceforth referred to as overlap, non-overlap and top, indicating the perceived relationship between the constituent subunits. The overlap and non-overlap views are similar in composition to two defined views of the E. coli ribosome [15], and thus it is reasonable to retain the terminology. The top view has not previously been described in the literature. The most commonly occurring view in our population of 815 individual particles is the non-overlap view: classes 1-5 in Fig. 4, consisting of 46, 29, 31, 34 and 22 members, respectively. Although there were two other non-overlap classes determined, they were of high intraclass variance and contained only 16 and 17 members (data not shown). The small subunit is clearly recognisable lying to the right in lateral projection. Fig. 5 shows the constituent images comprising class 1. Although they appear similar to the eye, their sum shows structural detail far better.
Another commonly occurring view is the overlap view: classes 6 and 7 in Fig. 4, consisting of 35 and 32 members, respectively. Three other overlap classes were also determined in the analysis, but are not shown here since they consisted of only 17, 14 and 15 members. In studies of E. coli ribosomes [15-19], the stain excluding body on the left has been shown to represent the small subunit, with the large subunlt lying to the right and below. The least frequently occurring view is what we call the top view: classes 8 and 9 in Fig. 4, each consisting of 27 members. Another top class of 22 members, but with a high intraclass variance, is not shown. As in the non-overlap view, the profiles of the two subunits are distinct. In the top projection, we interpret the particle on the right to be the large subunit because it is the larger stain-excluding region and has a rounded hemispherical appearance. The particle on the left has a characteristic head and collar (in class 9) and a cylin-
264
Fig. 5. The 46 individualimagescomprisingoverlap class 1. Their averageis image 1 in Fig. 4.
drical profile, suggesting that it is the small subunit. Reflecting the top view through a vertical axis brings the two subunits into the same relative positions as in the non-overlap view. This mirrored top view then appears to differ from the non-overlap projection by a rotation about an axis passing through the two subunits and in the plane of the image. Unfortunately, we were unable to prove these conjectures using angular reconstitution techniques [20]. The total numbers of complexes lying in the nonoverlap, overlap and top orientations is 195 (24% of 815), 113 (14%) and 76 (9%), respectively. The relative proportions of non-overlap/overlap/top views is thus 51:29:20. The non-overlap projection has the lowest intraclass variance (Fig. 4), indicating it is a stable position in which the ribosome can adsorb to the carbon. The overlap and top projections have generally higher intraclass variances, perhaps indicating that they may 'rock' about a particular orientation.
Spatial resolution of characteristic views Visually, the quality of the class averages is much better than of the original images. An objective measure
of the resolution of detail in these images is obtained by the Fourier Ring Correlation method [6]. This method assesses the similarity between independent images by determining the cross-resolution, i.e., the value of the highest common spatial frequency present in the two images. Clearly, the comparison must be between similar image, either (a) independent class averages of the particle in the same orientation, (b) two 'half-averages' obtained by summing odd and even numbered members of a particular class or (c) any two members of a particular class. The cross-resolution between any two images comprising class 1 (Fig. 5) is approx. 7.5 nm. The two averages were formed by adding alternate class members, and the cross-resolution between the two sums was 1.8 nm, indicating the significant improvement attained by averaging. This figure is best interpreted as a resolution limit attained in this particular class. The reproducible spatial resolution between any two pairs of non-overlap views in Fig. 4 averages 3.7 nm, and ranges from 1.8-5.7 nm (there are 10 combinations of pairs). For both the overlap and top views, the calculated value for the reproducible spatial resolution is 3.9 nm.
265
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respectively. The non-overlap class occurs as a cluster, distinct from the rest of the population, at the top left of the plot. The overlap views form a relatively compact group at the top right, while the individual top views are more sparsely distributed in the central region of this space. The major group of images at the bottom of the plot in Fig. 6b consisted primarily of misaligned images; although these were clustered by the classification algorithm their classes were subsequently rejected from further consideration. The plots in Fig. 6 are defined only by two factorial coordinates, whereas 12 coordinates were used in image sorting. The compactness or sparseness of classes seen in the two-dimensional space should be interpreted with caution•
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Fig. 6. (a) Eigenimages 1, 2 and 3, representing the basis vectors corresponding to factorial coordinates 1, 2 and 3. These eigenimages account for 17.1, 6.3 and 3.3% of the total interimagevariancein the data set. (b) Plot of positions of 815 ribosomeimagesin a two-dimensional space representedby factorialcoordinates2 and 3. Each point represents 1 image•Plots of positions of imagescomprising(c) class 1 (non-overlap), (d) class 6 (overlap) and (e) class 8 (top). Meaning of eigenimages The first 12 eigenimages calculated during the multivariate statistical analysis accounted for 42.6% of the total interimage variance, while all 24 accounted for 53.7%. The hierarchical ascendant classification did not give significantly different results if 12 or 24 factorial coordinates were used for the clustering; the results presented here were obtained by grouping in a 12-dimensional space. The first eigenimage (Fig. 6a) points to the center of mass of the entire data set, and resembles the predominant (non-overlap) projection. The second eigenimage appears to define the difference between the non-overlap and other views, especially with respect to the stain-excluding region occupied by the small subunit, and the off-center stain pit. The third eigenimage characterises less easily described differences in ribosome size, shape and internal stain distribution. A plot of the positions of all 815 images in a two-dimensional space, defined by factorial coordinates 2 and 3, is shown in Fig. 6b. Three major groupings are suggested: at the top left, the top right, and the bottom of the plot. Figs. 6c-e show only the images comprising class 1 (non-overlap), class 6 (overlap) and class 8 (top),
Comparison with E. coli 70 S monosome structure The 70 S ribosome of E. coli was surveyed in the electron microscope by Lake [15]. Two main views of the particle were distinguished, called overlap or nonoverlap, corresponding to whether the profiles of the large and small subunits overlapped. Boublik et al. [16] investigated technical factors of specimen preparation, but analysed only one particular view corresponding to Lake's overlap view. The relative orientation of the two subunits was examined by immunoelectron microscopy [17,18]• More recently, Verschoor et al. [19] applied image sorting and averaging algorithms to electron images of E. coli 70 S ribosomes. It is worth describing their analysis technique in detail, since it differs significantly in many respects from our own work here. Verschoor and colleagues studied electron micrographs of 70 S particles from E. coli using a double-carbon 'sandwich' preparation [2,16]. They selected a total of 308 individual particles, classified these visually into L, O and R types (73, 89 and 146 particles, respectively), and formed averages of the subsets. The L type is the non-overlap view, while the O and R types correspond roughly to Lake's original overlap view. Using a combination of principal components analysis (with only eight factorial coordinates) and electron micrographs of tilted specimens, it was shown that the O and R types related to endpoints of a continuum of rotation-related views spanning about 50 degrees. These results were later confirmed by Frank et al. [21] using a hybrid clustering algorithm. In this paper, we describe an analysis of electron micrographs of Th. lanuginosus ribosomes, negatively stained and lying on a single carbon layer• The ribosomes from this organism appear similar in outline and internal staining pattern to those from the eubacterium E. coli, although the rRNA compositions are different. Th. lanuginosus ribosomes adsorb to the grid in one of three preferred ways, giving three distinct projections; two of these correspond to previously studied adsorption modes for E. coli ribosomes. The relative propor-
266 tions of complexes seen in different views are significantly different for the two species; the non-overlap form occurs most frequently for the thermophilic fungus, whereas the overlap form is commonest for the eubacterium. It was not possible to distinguish subsets of overlap views of Th. lanuginosus ribosomes differing by a single axis rotation, because of the relatively small number of particles lying in this position, as well as the limited spatial resolution of the images used in this study. The Th. lanuginosus ribosomes also lie on the carbon film in another orientation, the top view, which appears to be related to the non-overlap view by a rotation about an axis perpendicular to the long axes of the constituent subunits. This projection form occurs infrequently, although it has been observed in analyses of large data sets of E. coli ribosomes (Boekema, E., Harauz, G. and Van Heel, M., unpublished data). Perhaps the key factors to its elucidation were: (i) the use of a single carbon preparation [4,16] and (ii) the relatively large size of the data set subjected to multivariate statistical analysis.
Comparison with other eukaryotes and thermophiles Eukaryotic ribosomes have not been studied by electron microscopy to as great an extent as those from prokaryotes, with emphasis having been on subunit structure [22,23]. Ribosomes from the vegetative amoebae and spores of the slime mold Dictyostelium discoideum have been imaged, but sophisticated image processing techniques were not available at that time [24]. The D. discoideum ribosomes were about 26 nm in size and clear non-overlap views were distinguished. Other studies of three-dimensional reconstructions of ribosomes from chick embryos to 5.5 nm resolution enabled the localisation of the exit channel for nascent protein, but these images cannot be directly compared with those of the Th. lanuginosus ribosomes derived here [25]. The only statement that can be made at present is that the mutual orientations of the large and small subunits in eubacterial and eukaryotic ribosomes are the same. The structures of ribosomal subunits from one other thermophilic organism, the eubacterium Bacillus stearothermophilus, have been studied [7,13]. This organism grows optimally at 65°C. Although subtle differences when compared to E. coli ribosomes were detected, it is not clear how these morphological alterations are related to thermophily. It is perhaps not surprising that ribosomes from a moderate thermophile may have even less easily detectable structural modifications than those from an extreme one. In general, though, macromolecular complexes isolated from thermophiles are better suited for structural studies, forming crystals in a time span in which their mesophilic counterparts are degraded [13,26]. Moreover, since con-
formational differences in functionally or compositionally distinct ribosomes may be less than 1 nm in size (e.g., Ref. 24 and 27), a higher resolution study is required. This may involve processing of electron images of unstained specimens, or attempts at crystallisation and X-ray diffraction studies. Our immediate future investigations on Th. lanuginosus ribosomes will include separation and analysis of the individual subunits to higher resolution by imaging at a higher magnification, and three-dimensional reconstruction from the characteristic projection views using micrographs of tilted specimens [3,28]. This approach will help to determine accurately the relative orientations between the different classes.
Conclusions Ribosomes from the thermophile Th. lanuginosus have been examined by electron microscopy and image analysis of negatively stained preparations. The complexes were about 24.5-26.5 nm in size, and 3 predominant projection forms were constructed to a reproducible spatial resolution approaching 1.8 nm. The characteristic projections of Th. lanuginosus ribosomes are similar in gross appearance to those described in the literature from the eubacterium E. coli, but occur in different proportions. This reflects the complexes' protein and rRNA compositions, which affect their adsorption behaviour. Despite the evolutionary distance between these two organisms, their differing protein and rRNA content, and the proven ability of our algorithmic approach to demonstrating subtle differences in structural features of ribosomes [4], quaternary ribosome structure is highly conserved, indicating its important relation to ribosome function.
Acknowledgements This work was supported by grants from the Research Advisory Board of the University of Guelph, and the Natural Sciences and Engineering Research Council of Canada to G.H. We are grateful to Dr. Patrick Whippey (University of Western Ontario) for assistance with densitometry, to Dr. Dawn Larson and Dr. Peter Zahradka for their comments on the manuscript, and to a reviewer for comments on the interpretation of the top view.
References 1 Wittmann, H.-G. (1983) Annu. Rev. Biochem. 52, 35-65. 2 Stoeffler-Meilicke, M. and Stoeffler, G. (1988) Methods Enzymol. 164, 503-520. 3 Frank, J., Radermacher, M., Wagenknecht, T. and Verschoor, A. (1988) Methods Enzymol. 164, 3-35. 4 Harauz, G., Boekema, E.J. and Van Heel, M.G. (1988) Methods Enzymol. 164, 35-49.
267 5 van Heel, M. (1984) Ultramicroscopy 13, 165-183. 6 Van Heel, M. (1987) Ultramicroscopy 21, 95-100. 7 Van Heel, M. and Stoeffler-Meilicke, M. (1985) EMBO J. 4, 2389-2395. 8 Harauz, G., Stoeffler-Meilicke, M. and Van Heel, M. (1987) J. Mol. Evol. 26, 347-357. 9 Domsch, K.H., Gains, W. and Anderson, T.-H. (1980) Compendium of Soil Fungi, Academic Press, New York. 10 Nazar, R.N. and Wildeman, A.G. (1983) Nucleic Acids Res. 11, 3155-3168. 11 Nazar, R.N., Wong, W.M. and Abrahamson, J.L.A. (1987) J. Biol. Chem. 262, 7523-7527. 12 Miller, H.M. and Shepherd, M.G. (1973) Can. J. Microbiol. 19, 761-763. 13 Yonath, A. and Wittmann, H.-G. (1988) Methods Enzymol. 164, 95-107. 14 Van Heel, M. and Keegstra, W. (1981) Ultramicroscopy 7, 113130. 15 Lake, J.A. (1976) J. Mol. Biol. 105, 131-159. 16 Boublik, M., Hellmann, W. and Kleinschmidt, A.K. (1977) Cytobiologie 14, 293-300.
17 Kastner, B., Stoeffler-Meilicke, M. and Stoeffler, G. (1981) Proc. Natl. Acad. Sci. USA 78, 6652-6656. 18 Lake, J.A. (1982) J. Mol. Biol. 161, 89-106. 19 Verschoor, A., Frank, J., Wagenknecht, T. and Boublik, M. (1986) J. Mol. Biol. 187, 581-590. 20 Van Heel, M. (1987) Ultramicroscopy 21, 111-124. 21 Frank, J., Bretaudiere, J.-P., Carazo, J.-M., Verschoor, A. and Wagenknecht, T. (1988) J. Microsc. 150, 99-115. 22 Frank, J., Verschoor, A. and Boublik, M. (1981) Science 214, 1353-1355. 23 Lutsch, G. and Bielka, H. (1988) Eur. J. Biochem. 172, 653-662. 24 Boublik, M. and Ramagopal, S. (1980) Mol. Gen. Genet. 179, 483-488. 25 Milligan, R.A. and Unwin, P.N.T. (1986) Nature 319, 693-695. 26 Morikawa, K., Fujiyoshi, Y., Ishizuka, K., Sugiyama, J., Kawakami, M. and Takemura, S. (1986) J. Microsc. 142, 247-258. 27 Vasiliev, V.D., Selivanova, O.M., Baranov, V.I. and Spririn, A.S. (1983) FEBS Lett. 155, 167-172. 28 Wagenknecht, T., Radermacher, M. and Frank, J. (1987) in Fifth Conversation in Biomolecular Stereodynamics, pp. 251-252, Adenine Press, New York.
