Structural study of tetragonal-ordered aggregates of phage φ29 necks

JOURNAL OF ULTRASTRUCTURE RESEARCH

89, 79-88 (1984)

Structural Study of TetragonaI-Ordered Aggregates of Phage 429 Necks J o s g M . CARAZO,* NARCISO G A R C I A , t ANTONIO SANTISTEBAN,t AND JOSI~ L. CARRASCOSA*

*Centro de Biologia Molecular, CSIC, Universidad Aut6noma, 28049 Madrid, and ~flBM Scientific Centre, Paseo Castellana 4, 28046 Madrid, Spain Received May 22, 1984, and in revisedform August 15, 1984

A new class of two-dimensional tetragonal aggregates of phage ¢29 necks has been studied by electron microscopy and a combination of Fourier filtering procedures and detailed rotational analysis. The results confirm the main features of the head-to-tail connectingregion previously observed in hexagonalaggregates. There are several differencesin the resulting pictures that can be attributed to the different way in which the aggregates are organized and stained. © 1984 AcademicPress,Inc.

The head-to-tail connecting region of complex bacteriophages is located in an unique vertex of the icosahedral head. This region presents a structure called the connector that has been involved in prohead assembly and DNA packaging (Camacho et al., 1977; Hendrix, 1978; Hsiao and Black, 1978; Murialdo and Becker, 1978; Van Driel and Couture, 1978; Kochan and Murialdo, 1983; Nakasu et al., 1983). Phage connectors have been purified and biochemically analyzed (Tsui and Hendrix, 1980; Matsuo-Kato et al., 1981), but only in a few cases a detailed structural analysis o f the connectors has been carried out. Driedonks et al. (1981) presented an image of T4 in vitro assembled connectors based on rotational analysis. Carrascosa et al. (1982, 1983) studied the neck of phage ~b29, purified from phage particles by Fourier and rotational filtering of two-dimensional hexagonal aggregates. Kochan et al. (1984) have studied the connector of phage X by optical filtering o f two-dimensional hexagonal aggregates. The resulting pictures of these three structures are very similar, both in dimensions and in their main morphological features. Nevertheless, it must be noticed that the crystalline arrangement by itself can influence the way in which individual specimens are negatively stained, by rendering

the different domains of the specimen more or less accessible to the stain. The safest way to validate the results attained by Fourier image filtering techniques is the obtention of similar results in two different types of regular aggregates of the same individual structure. If the filtering procedure applied to both types of aggregates yields equal results, the existence of artifacts can be discarded. This kind of validation has been carried out in the structural studies of projections of fibrinogen crystallized in the symmetry groups P21212 and P21 (Hewat et al., 1982), the membranebound acetylcholine receptor of Torpedo californica (Ross et al., 1977; Kistler and Stroud, 1981), and in three-dimensional reconstructions with the purple membrane in P3 (Henderson and Unwin, 1975) and P22121 (Leifer and Henderson, 1983). A similar validation for the structure o f the neck of bacteriophage ~b29 has also been attained. Bacteriophage ~b29 is a doublestranded D N A virus that presents a complex structure. The viral particle is composed by only six structural proteins (Anderson et al., 1966; Mrndez et al., 1971; Carrascosa et al., 1981). Two of these proteins, p l 0 and p 11, form the viral neck. The isolated necks can form in vitro two-dimensional hexagonal aggregates that have been studied pro-

79 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

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posing a structural model of the phage neck (Carrascosa et al., 1982, 1983). In this work we present the study of two-dimensional tetragonal aggregates of the same phage structures. The comparison between the neck pictures extracted from these two different aggregates has allowed us to obtain the main features of the neck particles with independence of the way in which they were arranged. MATERIALS AND METHODS

Crystal Preparation The head-to-tail connecting region of phage 4~29 can be purified from phage particles by chemical treatment (Carrascosa et aL, 1982). After purification, the phage necks have been induced to form hexagonal two-dimensional aggregates by drying over mica a concentrated preparation of necks mixed with negative stain (Home and Pasquali-Ronchetti, 1974; Carrascosa et al., 1982). In order to remove salts from neck preparations before crystallization, the samples were dialyzed against 0.1 M ammonium bicarbonate, and under these conditions most of the aggregates were hexagonal. If the necks were dialyzed against 0.1 M ammonium acetate, variable amounts oftetragonal aggregates also appeared. Both hexagonal and tetragonal aggregates could be more easily produced if the spreading and drying over mica were done in the presence of 25% ammonium sulfate.

Electron Microscopy The neck-containing samples dried over mica were coated with a thin layer of carbon and floated over 2% uranyl formate. The sample containing film was picked up with carbon-coated grids. Shadowed crystals were obtained by mixing the phage necks with the negative stain and the ammonium sulfate, as described before. After extension over mica and air-drying, the samples were shadowed with platinum--carbon (30 ~) with an elevation of 20 °. A carbon film (20 ~) was deposited to stabilize the platinum film, and the replica was floated on double-distilled water and picked with electron microscope grids. Samples were photographed in a JEOL 100B electron microscope equipped with an anticontamination device. The magnification was calibrated using catalase crystals as an internal standard (Wrigley, 1968). Electron micrographs were analyzed by obtaining their diffraction patterns in a folded-beam optical bench. Only those micrographs with negligible astigmatism and presenting sharp spots in the diffraction pattern extending to high resolution were selected for digital image processing.

Image Processing Selected micrographs were digitized in a Perkin-Elmer 1010A flat-bed microdensitometer using a sampling interval of 20 gm corresponding to 0.5 nm on the specimen and a scale linear in optical density. (a) Fourier analysis and filtering. The digital twodimensional Fourier filtering was performed using the spatial filtering approach proposed by Garcla et al. (1981) and Carrascosa et al. (1982). The bending of the areas to be processed was corrected by a geometric transformation, mad the images were then resampled in order to make their Fourier transform match a perfect predefined lattice-pass filter in the frequency domain. The inverse Fourier transform was computed from the above selected lattice spots without any assumption about point symmetries, thus obtaining the final Fourier-filtered image. To know the degree of spatial ordering, as well as the possible axial symmetries present in the q~29 neck aggregates, two parameters were obtained from their Fourier transforms: The degree of spatial ordering of the aggregates was studied calculating the total figure of merit, which expresses the consistency of the phase (Ross et al., 1977). The existence of axial symmetries was studied computing the minimum phase residual for several point symmetries. (b) Rotational analysis and filtering. Two different motifs composed the crystal unit cell. Both motifs were extracted from each filtered crystal in order to study their internal structure. Two types of information were obtained from their analysis. The first one was the power spectra, which measures the relative weight of each harmonic in the Fourier series expansion of the image in the angular polar coordinate (Crowther and Amos, 1971). The second one was the relative phase difference of each harmonic component with respect to a fixed space direction (the origin of the angular coordinate); this indicates the relative axial orientation of different harmonics in the same or in different images. In the latter case, much care was taken to assure that the reference space direction was the same for all images. It is important to notice that relative phase differences are meaningful only in the interval (0°, 360°/ n), with n being the harmonic under study, for they repeat outside this interval. The averaged picture of both motifs was obtained from the best four Fourier-filtered images. The averaging procedure was based on the existence of a significant amount of 6-fold symmetry in one of the two motifs. Crystal images were rotated by an angle of 90 ° when it was required to get a good match of the 6th harmonic component of the motif that showed a clear 6-fold symmetry, obtaining correlations higher than 0.95 among these components. Crystal images were then averaged, and the power spectra of both averaged motifs calculated. These averaged motif images were rotationally filtered. The filtered images were constructed by the ad-

STRUCTURAL STUDY OF PHAGE 4~29 NECKS

81

FIG. 1. A tetragonal neck aggregatenegatively stained with uranyl formate. The dimensions of the aggregates were variable. This image shows a large ordered area.

dition of the 0th (mean radial intensity), 6th, and 12th harmonic components. RESULTS

Crystal Characterization Purified necks o f phage q529 f o r m ordered aggregates w h e n they dry o v e r mica. T h e nature o f the order in the aggregates was found to d e p e n d on the buffer conditions in which the sample was stored before drying (see Materials and Methods). T w o types o f t w o - d i m e n s i o n a l lattices were found. One lattice type was hexagonal (Carrascosa et al., 1982, 1983), a n d the other tetragonal. T h e tetragonal t w o - d i m e n s i o n a l aggregates o f q529 necks were irregular in shape and size (dimensions ranged f r o m a b o u t 50 to 10 000 n m ) (Figs. 1, 2). A total o f 13 tetragonal aggregates were selected for image processing. In two cases, aggregates were big enough to allow the selection o f two different areas in the s a m e

crystal to be processed, giving a total o f 15 processed areas. A careful e x a m i n a t i o n o f their c o m p u t e d Fourier t r a n s f o r m (Fig. 3) showed a tetragonal lattice f o r m e d by strong spots, but with s o m e weak extra-spots located at the centers o f s o m e squares defined by those strong spots. This fact could be interpreted assuming that the real tetragonal lattice existing in the crystal was the one f o r m e d by the strong and the weak spots, or that the weak extra-spots were just noise. Nevertheless, this feature was reproducible in all the areas studied, suggesting that the weak spots were, in fact, not noise. T h e Fourier t r a n s f o r m then showed a tetragonal lattice with a - - b = 16.5 +_ 0.8 nm, ~ = 90 +_ 2 ° ( m e a s u r e m e n t s f r o m 40 aggregates, including not digitally processed micrographs). T h e spots extended to 1/3 n m in the reciprocal space. T h e values obtained for the total figure o f m e r i t ranged f r o m 0.5 to 0.6 up to the

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sixth order in the areas processed. These values indicated that the spatial ordering o f the tetragonal neck aggregates was not very good. As a c o m p a r i s o n , in the case o f the hexagonal aggregates that figure ranged f r o m 0.7 to 0.8 (closer to 1.0, the ideal case) up to a similar resolution. These results indicated that the hexagonal a r r a n g e m e n t o f necks was better ordered t h a n the tetragonal one.

Fourier Filtering T h e m o s t relevant features on the Fourier-filtered images were: 1. T w o different types o f necks were present in the lattice (Figs. 4, 5). One was m o r e contrasted b y the negative stain than the other. In b o t h o f t h e m , there was a wellcontrasted inner region and a less contrasted

outer one (Figs. 4, 5). The overall m o r phology o f b o t h types o f necks was similar to the projected image obtained f r o m hexagonal aggregates (Carrascosa et al., 1982). The external region has been associated mainly with the connector protein p 10, while the internal one was p r o b a b l y built up by s o m e contribution o f p 10 a n d b y the other protein p l l , which f o r m s the lower collar observed in side views o f the neck (Carrascosa et al., 1982, 1983). 2. T h e distance between two necks o f the s a m e type was 16.5 __+ 0.8 n m (Fig. 4). T h e distance between necks o f different types (first neighbors) was 11.5 + 0.6 n m , smaller than the 15 + 1 n m obtained in the case o f hexagonal aggregates (Carrascosa et aL, 1982). This fact suggested that each type o f neck was in a different plane. This h y p o t h -

STRUCTURAL STUDY OF PHAGE 4~29NECKS

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FIG. 4. Fourier-filtered image from a tetragonal aggregate; the unit cell is shown. Two motifs forming the crystal unit cell are dearly visible. FIG. 5. Imageresulting from the addition of two separated Fourier-filtered subareas of the same tetragonal aggregate and the reinforcement of the P2 symmetry.

esis was confirmed by platinum shadowing tetragonal aggregates, which showed a clear tetragonal lattice (Fig. 6) with a lattice constant o f 17.0 + 0.8 nm, essentially equal to the distance between necks o f the same type in negatively stained aggregates (Fig. 7). This means that only one neck type was shadowed while both o f t h e m were negatively stained, indicating that the two types o f necks were at different levels. 3. The outer region o f b o t h types o f necks appeared as four defined pairs o f m o r p h o logical units connecting the adjacent necks and four less contrasted units (Figs. 4, 5). The difference in contrast suggested a certain overlapping o f pairs o f units from adjacent necks. Side views that could support this hypothesis have been actually found; Fig. 8 shows a side view o f a row o f collars with alternating orientation (schematically presented in Fig. 9) that overlapped in the region o f the upper collar o f the neck (connector protein p l 0 , Carrascosa et al., 1983). However, other spatial dispositions o f the necks cannot be excluded. 4. The inner and outer radii o f the well-

contrasted internal region were, in both types o f necks, very similar to those o f the 6-folded region o f the necks that form hexagonal aggregates (radii about 1.5 and 3.5 nm, respectively) (Carrascosa et al., 1982, 1983). As a matter o f fact, this region appeared slightly larger in the less contrasted neck type than in the m o r e contrasted one (see Figs. 4, 5). These data suggested that the region mainly built up by the protein p l 1 could be contrasted in two different ways, depending on the plane occupied by the necks in the tetragonal aggregate (Figs. 8, 9). One contrasting way was similar to the one that takes place in the hexagonal aggregates, while the other was different. The fact that the inner region was differently stained in both types o f necks led us to the rotational analysis o f this region, in order to get further insight into its structure.

Rotational Analysis and Filtering A rotational analysis o f 10 different specimens was carried out. The most significant features o f each neck type appeared enhanced in an average image obtained from

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® FIG. 6. 4~29 Neck aggregate shadowed with platinum--carbon at 20 ° inclination; the lattice vectors are indicated. Fie. 7, Negatively stained q~29 neck aggregate (same magnification as in Fig. 6) showing the lattice vectors. Note that the lattice vectors shown in this figure and in Fig. 6 are basically the same. FIG. 8. Side view o f a row o f negatively stained necks, obtained under conditions where tetragonal aggregates were formed. Note that necks seem to interact by their upper collar. F~o. 9. Schematic interpretation o f Fig. 8.

STRUCTURAL STUDY OF PHAGE q529 NECKS TABLE I ROTATIONAL POWER SPECTRAOF FILTERED q~29 NECKS More contrasted neck Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Energy (%) 21.92 1.80 28.27 2.85 4.80 17.88 4.83 16.00 0.32 0.24 0.15 0.03 0.07 0.03 0.05 0.01 0.04 0.04 0.02 0.01

Less contrasted n e c k Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Energy (%) 32.17 23.33 15.87 13.86 4.63 1.44 6.13 1.55 0.71 0.04 0.02 0.04 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01

Note. Necks of both staining types were extracted from four Fourier-filtered aggregates and averaged. Their rotational power spectra integrated between radii 3.1 and 4.2 nm clearly show a local maximum for the 6-fold component of the more-contrasted neck type and a local minimum for the same component of the less-contrasted neck type. A total of 50 rotational harmonics were obtained, though only the first 20 are shown.

the best four Fourier-filtered pictures. The most relevant results obtained about the inner region o f both types o f necks were: 1. The 6-fold c o m p o n e n t o f the more contrasted type o f necks, which appeared stained like the necks in hexagonal aggregates, presented a local m a x i m u m between radii 3.1 and 4.2 n m (the outermost part o f this internal region) (see Table I). The phase difference o f this c o m p o n e n t with reference to the origin o f the angular coordinate was nearly constant with the radius. This is what is expected to happen in a specimen with a significant 6-fold symmetry. 2. The 6-fold c o m p o n e n t o f the less con-

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trasted type o f necks presented a local mini m u m between radii 3.1 and 4.2 n m (Table I). The phase difference o f this c o m p o n e n t varied rather randomly with the radius. This situation could be the result o f a systematic annihilation o f the 6-fold component. (The most complete annihilation o f a n-fold c o m p o n e n t would come from the addition o f two equally weighted n-fold c o m p o n e n t s rotated by an angle o f 360°/2n, one with respect to the other.) In our case, the results would be explained if the 6-fold c o m p o n e n t o f the Fourier-filtered less contrasted neck picture were the result o f the addition o f 6-fold components, half o f them with a given axial orientation and the other half presenting a 30 ° rotation with respect to the former. The phase differences o f this 6-fold c o m p o n e n t had to be random, as it could be considered residual noise. The more contrasted neck type o f the above m e n t i o n e d average image was rotationally filtered (Figs. 10, 11). The filtered image was constructed by adding the 0th, 6th, and 12th h a r m o n i c c o m p o n e n t s o f its rotational series expansion (Figs. 11, 12), appearing very similar to the one observed in hexagonal aggregates (Fig. 13).

Crystal Symmetry Group Once we established the 6-fold s y m m e t r y o f one o f the differently stained types o f necks, we obtained the m i n i m u m phase residual assuming the crystal s y m m e t r y group P2 (Holser, 1958) for the tetragonal aggregates. This crystal s y m m e t r y group was the higher one compatible with a 6-folded m o t i f aggregated in a tetragonal lattice. T h e values obtained ranged from 33 to 50 °. As a comparison, in the case o f the hexagonal aggregates, assuming a s y m m e t r y group P6, the values ranged from 20 to 30 °. These results indicated that even a P2 s y m m e t r y was not clearly present in the majority o f the tetragonal aggregates. Only in small areas from two images the 6-fold c o m p o n e n t was significant in the inner region o f both types o f necks. The phase residuals obtained assuming a s y m m e t r y group P2 were 33 and 35 °

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FIG. 10. 429 Neck of the most contrasted type, obtained by averaging four different crystals. It shows a 6-fold symmetry in the inner region. FIG. 11. Rotational filtering of the image shown in Fig. 10. The 0th, 6th, and 12th rotational components have been used in this reconstruction. FIG. 12. Equallyspaced level curves of Fig. 11. F:G. 13. Equallyspaced level curves of the Fourier- and rotationally-filtered neck obtained from the hexagonal aggregates (Carrascosa et aL, 1982). The 0th, 6th, and 12th rotational components have been used in the reconstruction. in these two cases. These results suggested that in certain small areas the tetragonal ordering was almost o f the same quality o f the hexagonal ordering o f necks (Fig. 5). DISCUSSION We have studied the ~29 neck by image processing o f electron micrographs o f negatively stained two-dimensional tetragonal

aggregates. The resulting images have been c o m p a r e d to those obtained by Carrascosa et al. (1982, 1983) for the hexagonal aggregates formed by the same viral particles. The aim was to study the consistency o f its main structural features, as well as to get some insight into the m e c h a n i s m s o f negative staining. The unit cell o f the tetragonal aggregates

STRUCTURAL STUDY OF PHAGE 429 NECKS has been found to contain two differently stained types of necks. One neck type was more contrasted by the negative stain than the other. The more contrasted neck type appeared very similar to the neck picture obtained from the hexagonal aggregates. Its rotational power spectrum showed a clear 6-fold symmetry in the internal part, and a 12-fold symmetry in the external one. In the less contrasted neck type, a systematic annihilation of the 6-fold component was observed, remaining only the 12-fold symmetry in the external part. The 6-fold component of the more contrasted neck type showed a constant orientation in all aggregates.

Structural Model o f the Tetragonal Aggregates If the 4,29 necks had in fact the 6-fold and the 12-fold symmetries, the most plausible explanation of their arrangement into a tetragonal lattice would be that the interactions among first neighbor necks were driven mainly, although not exclusively, by the 12-folded part of the specimen, which is known to correspond mainly to the connector protein p 10 (Carrascosa et al., 1983). If the 6-folded part of the structure did not play any role in crystal formation, two possible orientations of the specimen could happen with the same probability, and the result would be a quasi-packaging instead of a true crystal. If the 6-folded part did play a role, the result would be a crystal with a P2 symmetry. The results obtained with the tetragonal aggregates are consistent with the following model. It is proposed that the necks of the more contrasted type would be oriented by interactions among the 12-folded and the 6-folded parts of the structure, and that the necks of the less contrasted type would be oriented almost solely by interactions among the 12-folded part of protein p 10, as seemed to be suggested by the disposition of the necks in Figs. 8 and 9. Every less contrasted neck could then rotate by 30 ° and still main-

87

tain the same interactions with its neighbors. This difference in the behavior of the two differently stained neck types could be related to their different relative positions with respect to the grid surface. Taking into account all these data, a comprehensive explanation of the rotational analysis can be given: The local maximum of the 6-fold component of the most contrasted neck type is produced by the averaging (during the Fourier filtering) of necks with the same orientation. The local minim u m of the same component of the less contrasted neck type is produced by the averaging of necks with a difference of orientation of 30 °. The uncertainty in the orientation of the less contrasted necks could account for the low values of both, the total figure of merit and the minimum P2 phase residuals (except for some small areas). Results lead us to suggest that these tetragonal aggregates of 4~29 necks are poor crystals. Our study has revealed that the neck, in spite of staining differences, presented the following main morphological features: 1. A hole in its center with about 3 nm of diameter. 2. An internal region well contrasted, with 6-fold symmetry, whose outer diameter ranged between 7 and 8 nm. 3. An external region built up by 12 morphological units with an outer diameter of about 13.5 nm. The fact that these same features are also observed in the hexagonal aggregates (in spite of their different staining and preservation conditions), indicates that these features of ~29 necks are actually reliable. Further details of their structure are presently being studied obtaining a three-dimensional reconstruction of the neck from the hexagonal aggregates. Taking into account the remarkable morphological similarities in the projected views of the structure of the phage head-to-tail connecting regions studied up to date (Driedonks et al., 1981; Carrascosa et al., 1982, 1983; Kochan et al., 1984), it is expected that the

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detailed study of their structure would help to understand their basic function mechanism. This work was partly supported by grants from Comisi6n Asesora para el Desarrollo de la Invesfigaci6n Cientifica y T6cnica, and Fondo de Invesfigaciones Sanitarias. REFERENCES ANDERSON, D. L., HICKMAN, D. D., AND REILLY, B. E. (1966) J. Bacteriol. 91, 2081-2089. CAMACHO,A., JIMI~NEZ,F., DE LA TORRE, J., CARRASCOSA, J. L., MELLADO, R. P., V,~ZQUEZ, C., VII~UELA, E., AND SALAS, M. (1977) Eur. J. Biochem. 73, 39-55. CARRASCOSA,J. L., MI~NDEZ, E., CORRAL, J., RUBIO, V., RAMiREZ, G., VII~UELA,E., AND SALAS, M. (1981) Virology 111, 401-413. CARRASCOSA, J. L., Vtg~UELA, E., GARCiA, N., AND SANTISTEBAN, A. (1982) J. Mol. Biol. 154, 311-324. CARRASCOSA,J. L., CARAZO, J. M., AND GARCIA, N. (1983) Virology 124, 133-143. CROWTHER, R. A., AND AMOS, L. A. (1971) J. MoL Biol. 60, 123-130. DRIEDONKS, R. A., ENGEL, g., TEN HEGGELER, B., AND VAN DRmL, R. (1981) J. Mol. Biol. 152, 641-662. GARC~AN., SANTISTEBAN,A., AND CARRASCOSA, J. L. (1981) Proc. 2nd Scand. Conf. Image Analysis. Helsinki, pp. 444-449. HENDERSON, R., AND UNWIN, P. N. T. (1975) Nature (London) 257, 23-32. HENDRIX, R. W. (1978) Proc. Natl. Acad. Sci. USA 75, 4779-4783.

HEWAT, E. A., TRANQUI,L., AND WADE, R. H. (1982) J. MoL Biol. 161, 459-477. HOLSER, W. T. (1958) Z. KristaUogr. 111, 266-281. HORNE, R. W., AND PASQUALI-RONCHETTI,I. (1974) J. Ultrastruct. Res. 47, 361-383. HSIAO, C. L., AND BLACK, L. (1978) Virology 91, 2638. JIMI~NEZ,J., AND NAVALdlN, J. L. (1982) I B M J . Res. Dev. 26, 724-734. KOSTLER, J., AND STROUD, R. M. (1981) Proc. Natl. Acad. Sci. USA 78, 3678-3682. KOCHAN, J., CARRASCOSA, J. L., AND MURIALDO, H. (1984) J. Mol. Biol. 174, 433-447. KOCHAN, J., AND MURIALDO,H. (1983) Virology 131, 100-115. LEIFER, D., AND HENDERSON,R. (1983) J. Mol. Biol. 163, 451-466. MATSUO-KATo,H., FUJISAWA,H., AND MINAGAWA,T. (1981) Virology 109, 157-164. MI~NDEZ, E., RAMiREZ, G., SALAS,M., AND VINUELA, E. (1971) Virology 45, 567-576. MURIALDO, H., AND BECKER,A. (1978) Microbiol. Rev. 42, 529-576. NAKASU, S., FUJISAWA,H., AND MINAGAWA,T. (1983) Virology 127, 124-133. Ross, M. J., KLYMKOWSKY, M. W., AGARD, D. A., AND STROUD, R. M. (1977)J. Mol. Biol. 116, 635-659. TsuI, L., ANDHENDRIX,R. W. (1980)J. Mol. Biol. 142, 419-438. VAN DRIEL, R., AND COUTURE, E. (1978) J. Mol. Biol. 123, 115-128. WRIGLEY, N. G. (1968) J. Ultrastruct. Res. 24, 454464.