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Visualizing the Secondary Structure of Tubulin: Three-Dimensional Map at 4 Å
JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB973841
118, 119–127 (1997)
Visualizing the Secondary Structure of Tubulin: Three-Dimensional Map at 4 Å Eva Nogales, Sharon Grayer Wolf, and Kenneth H. Downing1 Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received October 7, 1996, and in revised form December 19, 1996
One of the characteristics of electron crystallography that sets it apart from X-ray crystallography is that structure factor phases can be determined with good accuracy directly from images of the specimens. This advantage allows density maps to be constructed at low to medium resolution in which certain features can be interpreted directly without the need for refinement based on an atomic model. On the other hand, collection of the data needed to construct a density map at sufficiently high resolution to build an atomic model is still a slow process. Thus it is natural that electron crystallographers try to extract the maximum amount of useful information from their reconstructions at each stage of the work, as the resolution improves. Maps generated from electron crystallographic data suffer from the problem that part of the data is inaccessible, because of the limitation on maximum specimen tilt in the electron microscope. The resulting ‘‘missing cone’’ of data produces an anisotropic point spread function, or blurring of the image in the direction perpendicular to the plane of the crystal. It has long been recognized that artifacts can be introduced in maps calculated at low to medium resolution from such limited data (Baumeister et al., 1986). On the other hand, when the resolution is sufficiently high, there is no ambiguity introduced by the anisotropy of the point spread function (Glaeser et al., 1989). We have been working toward a description of the structure of tubulin at atomic resolution, using electron crystallography of zinc-induced, two-dimensional sheets of the protein. The work has gone through several stages as data collection has progressed, allowing maps at successively higher resolutions to be reliably computed. In previous publications we presented three-dimensional reconstructions at a resolution of 6.5 Å (Nogales et al., 1995a; Wolf et al., 1996). Here we present a reconstruction at 4 Å, in which the greater detail invites further interpretation of the features seen in the map.
We are in the process of determining the structure of tubulin using electron crystallography of zincinduced, crystalline sheets. We have now extended the resolution to 4 Å, and there are many features in the map that appear to show details of the secondary structure. X-ray crystallographers are well aware of the problems of interpreting maps with such limited resolution, and the additional problem of the missing cone of data inherent in electron crystallography may make interpretation even more difficult. To investigate how reliably these maps can be interpreted, we have calculated density maps of a known structure, actin, under conditions similar to those of the tubulin map. Results of these simulations support the limited interpretations we made previously in the 6.5-Å maps and the more extensive interpretations we make here in the 4-Å map. Most of the secondary structure of the tubulin dimer can now be identified. r 1997 Academic Press INTRODUCTION
Solving the structure of proteins is today considered a first important step toward understanding, and hopefully modifying, the function of these biomolecules. While X-ray protein crystallography is a mature and productive technique for obtaining highresolution data, and the first method of choice for structural biologists, electron crystallography is a developing alternative in those cases where twodimensional (2-D) crystals are easier to obtain than their 3-D counterparts (Baumeister and Typke, 1993). Electron crystallography has been successfully applied to membrane proteins, which naturally occur in a two-dimensional environment, and to a number of other proteins that can be induced to form monolayer crystals. 1 To whom correspondence should be addressed at 1 Cyclotron Road, M.S. Donner, Lawrence Berkeley National Laboratory, Berkeley, CA 94720. Fax: (510) 486 6488.
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1047-8477/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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These maps are in the resolution range where the deficiencies in the electron crystallographic reconstructions have not been thoroughly investigated. We have used the known X-ray structure of actin (Kabsch et al., 1990), another self-assembling cytoskeletal protein, to model the effects that limited resolution and anisotropy in the data have in the interpretability of the density map in terms of secondary structure. We find that while most alpha helices are well resolved at 6.5 Å, beta sheets are poorly resolved. When the resolution is extended to 4 Å, almost all of the secondary structures should be resolved. These results give us confidence in our interpretation of details in the improved tubulin map. Examination of the map allows us to account for most of the a-helical and beta sheet content expected from CD (circular dichroism) experiments and structure prediction methods, as well as some of the loops connecting these elements. ACTIN MODELS
In order to obtain an idea of the relation between the density maps at 6.5 and 4 Å, and the actual structure, we calculated maps of actin under conditions designed to simulate the conditions of the
tubulin data. While there is no homology between actin and tubulin, they both contain a mixture of a-helix and beta sheet structures without a preferential orientation of the secondary structure elements. In contrast, the membrane proteins solved to this day by electron crystallography are dominated by a helices oriented roughly perpendicular to the crystal plane. Both actin and tubulin self-assemble into cytoskeletal fibers that interact with motor proteins, so they are of interest to overlapping research fields. The actin atomic coordinates from the Brookhaven Protein Data Bank were used with the program SFALL from the CCP4 crystallographic computing suite (Collaborative Computational Project, 1994) to compute the maps. Structure factors were modified in two ways to correspond to the experimental data. First, data were eliminated from a cone of half angle 30°, to match the missing cone of electron microscope data. The direction of the missing cone is identified as the c-axis and is indicated in Fig. 1. Second, a temperature factor was applied in the direction of this cone that reduced the amplitudes by a factor of four at 4 Å. Figure 2 shows the change in appearance of two a helices in the actin structure as the resolution is
FIG. 1. Ribbon diagram of actin (Kabsch et al., 1990). The arrow indicates the direction of the c-axis used for the simulation. Secondary structure elements referred to in the text and in following figures have been arbitrarily labeled.
TUBULIN SECONDARY STRUCTURE
reduced and anisotropy introduced. Figures 2a and 2b show the backbone (yellow line) as well as the 2.1-Å electron density (brown) of Helix A (see Fig. 1 to identify helices and beta sheets of Figs. 2 and 3 within the actin structure). The white surface in (a) corresponds to this area of the protein after limiting the resolution to 6.5 Å and applying a missing cone in the direction of the red lines, as previously described. The helix appears as a well-resolved cylindrical density, although the ends are not well defined. At 4 Å (Fig. 2b) turns can clearly be distinguished as well as the presence of larger residues. Similar effects can be seen in Figs. 2c and 2d for helix B. In this case, most probably due to the different orientation with respect to the c-axis, the helix is not as well defined. The presence of large aromatic residues can also contribute significantly to the appearance of continuity of helices at 4 Å. For helix C, one of the regions in the actin molecule with a higher abundance of bulky residues, the effect of the limited resolution and the missing cone is to overemphasize the side chains with respect to the backbone that thus appears almost discontinuous (not shown). Figure 3 shows the effect of the reduced resolution for two of the actin beta sheet regions. At 6.5 Å the sheets appear as very low densities compared with a helices. They only become visible at a very low isosurface level, at which they appear as discontinuous flat slabs of density (a and c). At 4 Å, however, the beta strands are some of the most prominent and easily interpretable features. Not only are the strands very clear, but the presence of large residues can also be detected (b and d). TUBULIN 3-D RECONSTRUCTIONS
The procedures that we are using in the electron crystallographic study of tubulin have been described previously, along with the earlier reconstructions (Nogales et al., 1995a, b; Wolf et al., 1996). The current data set includes 149 images, 115 of which were of specimens tilted to between 45° and 60°. A
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set of 30° images used in computing the first map, as well as some of the earlier 45° images, have been removed since their quality was not as high as that of the more recent data. Most of the newer images are at tilt angles of 55°–60°, greatly reducing the anisotropy of the map. A summary of phase residual statistics is given in Table 1. The sampling of data along reciprocal lattice lines is now highly redundant out to intermediate resolution. At the upper end of the current resolution limit of 3.5 Å, the data have not yet been so completely sampled, though they are good enough to make a 4-Å map highly reliable. Thus in the present report, we have limited the map resolution to 4 Å. Higher resolution maps have been generated and show a tantalizing increase in detail, but with a corresponding increase in the apparent noise level. Structure factor amplitudes have been obtained from a set of 93 diffraction patterns, 65 of them of specimens tilted 45°–60°. The average Rmerge when merging the patterns to a resolution of 4 Å was 23.8%. Figure 4 is a representation of parts of these data, where the intensity is shown along three planes that slice through the 3-D intensity data. The horizontal plane (A), parallel to the sheet, displays the intensity distribution seen earlier in diffraction patterns from untilted specimens (Downing and Jontes, 1992; Nogales et al., 1995b). Spacings around 4.8 Å are prominent, running along the protofilament direction, along with spacings around 10 Å at about 45° to the protofilaments. Especially at low resolution, intensities on odd rows of spots perpendicular to the protofilaments are weak, reflecting the similarity of the secondary structure in the a and b monomers. At higher resolution, differences in the amino acid sequences become more important in the diffraction intensities, and the odd rows are substantially stronger. In vertical section B, the 4.8-Å spacings, corresponding mainly to beta sheet interstrand spacings, are seen to be confined to a region near the central plane. The 10-Å spacings, interpreted as
FIG. 2. Actin region corresponding to helices A (a and b) and B (c and d) (see Fig. 1) shown at different resolutions (sampling of 1 Å/pixel). The backbone of the helices is shown in yellow. The electron density at 2.1 Å, calculated with the full data, is shown in brown. The white surface in (a) and (c) shows the effect of a 6.5-Å resolution cutoff, a cone of missing data, and loss of signal parallel to the c-axis. The effect of a 4-Å cutoff is shown in (b) and (d) in blue. The c-axis runs parallel to the red lines. FIG. 3. As Fig. 2 for beta sheets A (a and b) and B (c and d) (see Fig. 1). The isosurface for the 6.5-Å simulation (white surfaces) is much lower than that used in Fig. 2. FIG. 5. Three-dimensional reconstructions for a- and b tubulin at 4 Å (a) and 6.5 Å (b) (sampling of 1 Å/pixel). The bottom monomer is assigned to be the b subunit based on the presence of an extra density identified in a previous study as the taxol binding site (T) (Nogales et al., 1995a), which in turn has been shown to be in b tubulin (Rao et al., 1992; Combeau et al., 1994). The side of the molecules facing the viewer is presumed to correspond to the inside of the protofilament in the microtubule based on comparisons of our zinc-sheet reconstructions with lower resolution helical reconstructions of microtubules (Wolf et al., 1996). Microtubule reconstructions show a smooth ridge on the outside of the protofilament and a bumpy inside surface (Kikkawa et al., 1995). The red and blue lines on (b) indicate the approximate position of the sections shown in Figs. 6a and 6b. Elements identified in those sections have here been labeled to help the reader position them in the context of the whole molecule.
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FIG. 6. Different sections through the tubulin dimer model at 4 Å resolution. Blue and pink correspond approximately to a and b monomers, respectively. The translucent white surface shows the model at 6.5 Å. Color code of axes: green runs along protofilaments, red runs through the protofilament inside out (thus, it is the direction of the c-axis or missing cone), and blue runs across from one protofilament to another. The three sections were cut parallel to these axes. (a) Section cut near the region of contact between protofilaments, on the ‘‘taxol side’’ (see red line in Fig. 5b). This section includes three clear helical regions that are present in both a and b tubulin. They have been arbitrarily labeled H1, H2, and H3. The putative taxol site is labeled T. (b) Section parallel and contiguous to that in (a) showing what looks like a five-strand beta sheet (B1; see blue line in Fig. 5b). The individual strands have been arbitrarily labeled B1,1 to B1,5. The section also includes part of two helices, H4 and H5. The 6.5-Å surface is displayed at a much lower isosurface than in (a) and (c) in order to see density corresponding to the beta strands (see also the actin models above). (c) Section corresponding to part of the outside surface of the protofilament. The first three strands of B1 can be seen end-on, B1,3 connecting to H5. Two more long helices can be seen. H6 lies at the surface and crosses over a more internal helix, H7 (see also (d)). (d) Stereo view through the center of the b subunit showing beta sheet B1 and adjacent sheet, B2, running at an angle. The figure also shows an end-on view of H1, a tilted view of H2, and a side view of H3. Another short helix, labeled H8, is also shown end-on. The section includes part of the a subunit at the bottom, i.e., the corresponding H1. 123
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TABLE I Statistics for Image Data Set Resolution range (Å)
Number of reflections
Phase residual
100–14 14–7 7–5 5–4
3121 11 477 8594 7184
28.0° 19.8° 30.0° 36.7°
spacings between helices or sheets, increase in strength away from the plane. This is seen also in section C, which cuts parallel to the protofilaments. The distribution of diffraction intensities implies that the beta strands will be constrained to orientations nearly perpendicular to the crystal plane, with beta sheets mainly perpendicular to the protofilament axis. There is no clear indication of preferred orientation of a helices, although we may expect a tendency for features running in the directions of the diagonals of the unit cell. Figure 5a is an overall view of the 4-Å map. To help in orienting the new map with respect to those previously published, Fig. 5b shows a map built from the same data set but with a resolution cutoff at 6.5 Å. We note first that all of the major features of the new 6.5-Å map were already seen in the first map (Nogales et al., 1995a), although there is an improved sense of connectivity, with longer tube-like regions lining much of the outside surface (away from the viewer). The impression of the monomer outline has become more distinct. As displayed in Fig. 5, the 4-Å map is dramatically different from the 6.5-Å map, appearing at first as a collection of scattered, disconnected densities. Upon comparison with the lower resolution map, and more careful inspection, the relation between these densities and the anticipated secondary structure becomes clear throughout much of the map. The 6.5-Å map shows many cylindrical features that are highly suggestive of a helices. Several of these had been tentatively identified earlier, but the clarity in the present map is distinctly better. On the other hand, some of the regions previously interpreted as beta sheet are nearly invisible now at 6.5 Å at the isosurface shown. As seen in the actin simulations, the density in sheet regions is lower than in helices, so that a lower contour level must be used to see them. Beta sheets then appear as flat slabs (see Fig. 6b). The situation is quite different in the 4-Å map. Here most of the apparent helices can be seen as corkscrews with a clear righthand twist. The beta strands are particularly well defined, along with side chains sitting above and below the sheets. At this resolution, even some of the loop regions show up fairly distinctly.
Figure 6 shows different sections through the tubulin dimer2 reconstruction at 4 and 6.5 Å resolution, with main secondary structure elements labeled as helices (H) or beta strands (B). The three helices in Fig. 6a, H1, H2, and H3, can be very clearly seen for both a and b tubulins. At 6.5 Å they appear as smooth cylindrical surfaces, while at 4 Å the turns can be distinguished as well as some of the bulkier residues (e.g., see H1 in the b subunit). The appearance of the three helices at 4 Å is quite different due to their different orientations with respect to the c-axis (i.e., missing cone). While H1 is almost parallel to the c-axis, and can be seen end-on in the front view of Fig. 5, H3 is almost perpendicular to it and is shown in Fig. 5 approximately in a side view (see Fig. 6d for a clearer side view of H3). Helices perpendicular to the c-axis tend to appear as spring-like densities (see also H4, H5, H6, and H7), and those running parallel (H1 and H8) have a more compact appearance. The section shown in Fig. 6b corresponds to a beta sheet region (B1). The 6.5-Å white surface has been displayed at a much lower isosurface than in Figs. 6a and 6c in order to see density corresponding to the beta strands and appears as a discontinuous slab of density. Notice that the isosurface displayed in Fig. 5b lacks flat slabs, although helical regions are well represented. At 4 Å, however, individual strands are clearly visible. This is in good agreement with what we see in the actin simulations in the previous section. Individual strands are arbitrarily labeled in the figure. At its widest point (B1,3) the sheet runs through almost the whole depth of the molecule. Bulkier residues in the strands can be distinguished sticking toward and away from the viewer. The section also includes part of two helices that run around the sheet, perpendicular to the strands: H4, on the inside microtubule surface (front in Fig. 5), and H5, toward the outside surface. There is a very clear connection between B1,3 and H5, especially in the a subunit (see also Fig. 6c). A section corresponding to part of the putative outside surface of the protofilament (smoother surface, away from the viewer in Fig. 5) is displayed in Fig. 6c. This section shows the first three strands of B1 end-on, with B1,3 connecting to H5. There are two more helices, H6, which lies on the outside surface of the protofilament and crosses over a more internal helix, H7. Finally, Fig. 6d shows a stereo view through the center of the b-tubulin monomer
2 The definition of the dimer is still a matter of controversy. Given the similarity between the a–b and b–a longitudinal interactions it is difficult to assert which is the principal contact. We leave this point for further work.
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FIG. 4. Three-dimensional electron diffraction intensity data. Intensities are shown on three planes that cut through the data in three dimensions. Plane A is parallel to the plane of the crystal, with the protofilaments parallel to the direction marked h. Continuous intensities are seen along the reciprocal lattice lines in planes B and C.
showing beta sheet B1 and an adjacent sheet, B2, running at an angle. The figure also shows an end-on view of H1, a tilted view of H2, and a side view of H3, while H7 has been cut right through the middle. Another short helix, labeled H8, is also shown endon. The several large protrusions in this helix are most likely aromatic residues. The handedness of the reconstruction had been inferred previously by the assignment of the faces corresponding to inside and outside of the microtubule and by comparison with earlier low-resolution reconstructions (Wolf et al., 1996). At 4 Å resolution, the helices in this reconstruction are all righthanded (e.g., Fig. 6c), as is the twist of the beta
sheets (Fig. 6d), thus confirming that the hand has been presented correctly. In the 4-Å map, differences between the two monomers are dominated by densities that are presumably related to side chains and larger regions in the contact areas between monomers. There is an area of extra density in a tubulin at one end of helix H2 that is involved in the main lateral contact between protofilaments in the sheets. There is also an extra loop (or more ordered loop) visible in one of the longitudinal contacts, although it is difficult to decide whether it belongs to the a or b monomer. The putative taxol site (T in Figs. 5 and 6d) is the most prominent density in the b monomer that is not
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present in a. There is almost enough of a distinctive shape to this density to allow modeling of the orientation of the taxol molecule. DISCUSSION
Electron crystallography has been a fruitful tool for the study of tubulin structure, especially in the absence of high-resolution X-ray data. The methodology of electron crystallography has developed over the recent years to the point that it is possible to construct accurate atomic models of proteins from electron microscope data, but the progress in this work is still substantially slower than for an X-ray structure determination. The atomic model of bacteriorhodopsin (bR) (Henderson et al., 1990), the first determined by electron crystallography, required 15 years from the time that the first low-resolution structure was published, mainly because much of the technology had to be developed in the process. The light-harvesting complex (LHC) (Kuhlbrandt et al., 1994) was solved just 5 years after the first high-resolution projections were obtained, using the techniques that had been put in place by the previous work. We had expected that the tubulin structure would proceed even faster, given the advances in methodology in the last few years. However, the structure of tubulin is far more complex than that of bR. The basic 3-D structure of bR, seven transmembrane helices, was clear from the low-resolution map. The orientation of the helices, roughly perpendicular to the crystal plane, made it particularly easy to identify them with limited resolution. Only about a third of the tubulin structure is expected to be helix, and there is apparently no preferred direction with respect to the plane of the sheet. Extending the resolution of the bR map from 7 to 3.5 Å, even with a very anisotropic point spread function, allowed identification of sufficient structure within the helices to assign side chains to densities in the map. Most of the data for the bR structure were obtained from specimens tilted up to 45°, and the number of images was only about two-thirds of what we have in the tubulin data. Using electron diffraction amplitudes from specimens tilted up to 60°, the bR map has been substantially improved (Grigorieff et al., 1996). More of the loop regions are resolved, although the atomic model has very high temperature factors in nonhelical regions. The light-harvesting complex, on the other hand, contains a significant amount of loop within the molecule. Using a set of 79 images taken at tilts up to 60° the loop structure was better resolved than that in bR. Part of the improvement may have been a result of better sampling of the LHC data, due to the higher symmetry of the structure. Having P321
symmetry, the LHC has six monomers in the unit cell, compared to three for bR. Correspondingly, the zinc sheets have two tubulin heterodimers per unit cell, which has the effect of increasing the number of images required for the complete sampling of the data. The current data set for tubulin appears to be substantially more complete, and extends to higher angles, than that originally used for bR. The point spread function in our map is thus significantly more isotropic than in the bR map. This improvement in the data is reflected in the appearance of noticeably more detail, even in nonhelical regions, than in the bR map. The amount of detail that is already clearly visible in the present map is sufficient to allow us to begin the identification of specific secondary structure regions. Much of the molecule can now be seen in the 4-Å map to be composed of identifiable secondary structure. From secondary structure predictions and spectroscopic analyses we can expect on the order of 25% of the structure to be in sheet, 35% in helix, and the other 40% in loops, turns, and other structure (de Pereda et al., 1996). Regions identified to date would account for roughly 75% of the expected sheet and 90% of the helix. The secondary structure interpretation is now complete enough to allow us to begin searching protein structure databases to identify possible folding motifs within the tubulin structure. There are clear indications of densities for side chains in both helix and sheet regions in the map, and it should soon be possible to begin fitting the peptide chain to the map on the basis of the distribution of bulky side chains. Modeling some of the regions of the sequence that are strongly predicted to be helical should provide a firm start for the atomic model. Fitting the peptide will be helped by the specific similarities and differences between the a and b sequences and by the constraints of a growing body of biochemical, biophysical, and genetic data (Luduen˜a et al., 1992; Burns and Surridge, 1993; de Pereda et al., 1996). However, as expected at this resolution, the path of the backbone through loops connecting beta strands and a helices is not yet completely clear. Further extension of the resolution to 3.5 Å and addition of more data to improve sampling should resolve most of these unclear regions. REFERENCES Baumeister, W., Barth, M., Hegerl, R., Guckenberger, R., Hahn, M., and Saxton, W. O. (1986) Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans, J. Mol. Biol. 187, 241–253. Baumeister, W., and Typke, D. (1993) Electron crystallography of
TUBULIN SECONDARY STRUCTURE proteins: State of the art and strategies for the future, MSA Bull. 23, 11–19. Burns, R. G., and Surridge, C. D. (1993) Tubulin: Conservation and structure, in Hyams, J. S., and Lloyd, C. W. (Eds.), Microtubules pp. 3, 32 Wiley, New York. Collaborative Computational Project, N. 4. (1994) The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallogr. D50, 760–763. Combeau, C., Commerc¸on, A., Mioskowski, C., Rousseau, B., Aubert, F., and Goeldner, M. (1994) Predominant labeling of bover a-tubulin from porcine brain by a photoactivatable taxoid derivative, Biochemistry 33, 6676–6683. de Pereda, J. M., Leynadier, D., Evangelio, J. A., Chacon, P., and Andreu, J. M. (1996) Tubulin secondary structure analysis, limited proteolysis sites and homology to FtsZ, Biochemistry 35, 14203–14215. Downing, K. H., and Jones, J. (1992) Projection map of tubulin in zinc-induced sheets at 4 Å resolution, J. Struct. Biol. 109, 152–159. Glaeser, R. M., Tong, L., and Kim, S. H. (1989) Three-dimensional reconstructions from incomplete data: Interpretability of density maps at ‘‘atomic’’ resolution, Ultramicroscopy 27, 307–318. Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M., and Henderson, R. (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin, J. Mol. Biol. 259, 393–421. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman,
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E., and Downing, K. H. (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy, J. Mol. Biol. 213, 899–929. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Atomic structure of the actin:DNase I complex, Nature 347, 37–44. Kikkawa, M., Ishikawa, T., Wakabayashi, T., and Hirokawa, N. (1995) Three-dimensional structure of the kinesin headmicrotubule complex, Nature 376, 274–277. Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Atomic model of plant light-harvesting complex by electron crystallography, Nature 367, 614–621. Luduen˜a, R. F., Banerjee, A., and Khan, I. A. (1992) Tubulin structure and biochemistry, Curr. Opin. Cell Biol. 4, 53–57. Nogales, E., Wolf, S. G., Khan, I. A., Luduena, R. F., and Downing, K. H. (1995a) Structure of tubulin at 6.5 Å and location of the taxol-binding site, Nature 375, 424–427. Nogales, E., Wolf, S. G., Zhang, S. X., and Downing, K. H. (1995b) Preservation of 2-D crystals of tubulin for electron crystallography, J. Struct. Biol. 115, 199–208. Rao, S., Horwitz, S. B., and Ringel, I. (1992) Direct photoaffinity labeling of tubulin with taxol, J. Natl. Cancer Inst. 84, 785–788. Wolf, S. G., Nogales, E., Kikkawa, M., Gratzinger, D., Hirokawa, N., and Downing, K. H. (1996) Interpreting a mediumresolution model of tubulin: comparison of zinc-sheet and microtubule structure, J. Mol. Biol. 263, 485–501.
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Visualizing the Secondary Structure of Tubulin: Three-Dimensional Map at 4 Å Eva Nogales, Sharon Grayer Wolf, and Kenneth H. Downing1 Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received October 7, 1996, and in revised form December 19, 1996
One of the characteristics of electron crystallography that sets it apart from X-ray crystallography is that structure factor phases can be determined with good accuracy directly from images of the specimens. This advantage allows density maps to be constructed at low to medium resolution in which certain features can be interpreted directly without the need for refinement based on an atomic model. On the other hand, collection of the data needed to construct a density map at sufficiently high resolution to build an atomic model is still a slow process. Thus it is natural that electron crystallographers try to extract the maximum amount of useful information from their reconstructions at each stage of the work, as the resolution improves. Maps generated from electron crystallographic data suffer from the problem that part of the data is inaccessible, because of the limitation on maximum specimen tilt in the electron microscope. The resulting ‘‘missing cone’’ of data produces an anisotropic point spread function, or blurring of the image in the direction perpendicular to the plane of the crystal. It has long been recognized that artifacts can be introduced in maps calculated at low to medium resolution from such limited data (Baumeister et al., 1986). On the other hand, when the resolution is sufficiently high, there is no ambiguity introduced by the anisotropy of the point spread function (Glaeser et al., 1989). We have been working toward a description of the structure of tubulin at atomic resolution, using electron crystallography of zinc-induced, two-dimensional sheets of the protein. The work has gone through several stages as data collection has progressed, allowing maps at successively higher resolutions to be reliably computed. In previous publications we presented three-dimensional reconstructions at a resolution of 6.5 Å (Nogales et al., 1995a; Wolf et al., 1996). Here we present a reconstruction at 4 Å, in which the greater detail invites further interpretation of the features seen in the map.
We are in the process of determining the structure of tubulin using electron crystallography of zincinduced, crystalline sheets. We have now extended the resolution to 4 Å, and there are many features in the map that appear to show details of the secondary structure. X-ray crystallographers are well aware of the problems of interpreting maps with such limited resolution, and the additional problem of the missing cone of data inherent in electron crystallography may make interpretation even more difficult. To investigate how reliably these maps can be interpreted, we have calculated density maps of a known structure, actin, under conditions similar to those of the tubulin map. Results of these simulations support the limited interpretations we made previously in the 6.5-Å maps and the more extensive interpretations we make here in the 4-Å map. Most of the secondary structure of the tubulin dimer can now be identified. r 1997 Academic Press INTRODUCTION
Solving the structure of proteins is today considered a first important step toward understanding, and hopefully modifying, the function of these biomolecules. While X-ray protein crystallography is a mature and productive technique for obtaining highresolution data, and the first method of choice for structural biologists, electron crystallography is a developing alternative in those cases where twodimensional (2-D) crystals are easier to obtain than their 3-D counterparts (Baumeister and Typke, 1993). Electron crystallography has been successfully applied to membrane proteins, which naturally occur in a two-dimensional environment, and to a number of other proteins that can be induced to form monolayer crystals. 1 To whom correspondence should be addressed at 1 Cyclotron Road, M.S. Donner, Lawrence Berkeley National Laboratory, Berkeley, CA 94720. Fax: (510) 486 6488.
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1047-8477/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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These maps are in the resolution range where the deficiencies in the electron crystallographic reconstructions have not been thoroughly investigated. We have used the known X-ray structure of actin (Kabsch et al., 1990), another self-assembling cytoskeletal protein, to model the effects that limited resolution and anisotropy in the data have in the interpretability of the density map in terms of secondary structure. We find that while most alpha helices are well resolved at 6.5 Å, beta sheets are poorly resolved. When the resolution is extended to 4 Å, almost all of the secondary structures should be resolved. These results give us confidence in our interpretation of details in the improved tubulin map. Examination of the map allows us to account for most of the a-helical and beta sheet content expected from CD (circular dichroism) experiments and structure prediction methods, as well as some of the loops connecting these elements. ACTIN MODELS
In order to obtain an idea of the relation between the density maps at 6.5 and 4 Å, and the actual structure, we calculated maps of actin under conditions designed to simulate the conditions of the
tubulin data. While there is no homology between actin and tubulin, they both contain a mixture of a-helix and beta sheet structures without a preferential orientation of the secondary structure elements. In contrast, the membrane proteins solved to this day by electron crystallography are dominated by a helices oriented roughly perpendicular to the crystal plane. Both actin and tubulin self-assemble into cytoskeletal fibers that interact with motor proteins, so they are of interest to overlapping research fields. The actin atomic coordinates from the Brookhaven Protein Data Bank were used with the program SFALL from the CCP4 crystallographic computing suite (Collaborative Computational Project, 1994) to compute the maps. Structure factors were modified in two ways to correspond to the experimental data. First, data were eliminated from a cone of half angle 30°, to match the missing cone of electron microscope data. The direction of the missing cone is identified as the c-axis and is indicated in Fig. 1. Second, a temperature factor was applied in the direction of this cone that reduced the amplitudes by a factor of four at 4 Å. Figure 2 shows the change in appearance of two a helices in the actin structure as the resolution is
FIG. 1. Ribbon diagram of actin (Kabsch et al., 1990). The arrow indicates the direction of the c-axis used for the simulation. Secondary structure elements referred to in the text and in following figures have been arbitrarily labeled.
TUBULIN SECONDARY STRUCTURE
reduced and anisotropy introduced. Figures 2a and 2b show the backbone (yellow line) as well as the 2.1-Å electron density (brown) of Helix A (see Fig. 1 to identify helices and beta sheets of Figs. 2 and 3 within the actin structure). The white surface in (a) corresponds to this area of the protein after limiting the resolution to 6.5 Å and applying a missing cone in the direction of the red lines, as previously described. The helix appears as a well-resolved cylindrical density, although the ends are not well defined. At 4 Å (Fig. 2b) turns can clearly be distinguished as well as the presence of larger residues. Similar effects can be seen in Figs. 2c and 2d for helix B. In this case, most probably due to the different orientation with respect to the c-axis, the helix is not as well defined. The presence of large aromatic residues can also contribute significantly to the appearance of continuity of helices at 4 Å. For helix C, one of the regions in the actin molecule with a higher abundance of bulky residues, the effect of the limited resolution and the missing cone is to overemphasize the side chains with respect to the backbone that thus appears almost discontinuous (not shown). Figure 3 shows the effect of the reduced resolution for two of the actin beta sheet regions. At 6.5 Å the sheets appear as very low densities compared with a helices. They only become visible at a very low isosurface level, at which they appear as discontinuous flat slabs of density (a and c). At 4 Å, however, the beta strands are some of the most prominent and easily interpretable features. Not only are the strands very clear, but the presence of large residues can also be detected (b and d). TUBULIN 3-D RECONSTRUCTIONS
The procedures that we are using in the electron crystallographic study of tubulin have been described previously, along with the earlier reconstructions (Nogales et al., 1995a, b; Wolf et al., 1996). The current data set includes 149 images, 115 of which were of specimens tilted to between 45° and 60°. A
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set of 30° images used in computing the first map, as well as some of the earlier 45° images, have been removed since their quality was not as high as that of the more recent data. Most of the newer images are at tilt angles of 55°–60°, greatly reducing the anisotropy of the map. A summary of phase residual statistics is given in Table 1. The sampling of data along reciprocal lattice lines is now highly redundant out to intermediate resolution. At the upper end of the current resolution limit of 3.5 Å, the data have not yet been so completely sampled, though they are good enough to make a 4-Å map highly reliable. Thus in the present report, we have limited the map resolution to 4 Å. Higher resolution maps have been generated and show a tantalizing increase in detail, but with a corresponding increase in the apparent noise level. Structure factor amplitudes have been obtained from a set of 93 diffraction patterns, 65 of them of specimens tilted 45°–60°. The average Rmerge when merging the patterns to a resolution of 4 Å was 23.8%. Figure 4 is a representation of parts of these data, where the intensity is shown along three planes that slice through the 3-D intensity data. The horizontal plane (A), parallel to the sheet, displays the intensity distribution seen earlier in diffraction patterns from untilted specimens (Downing and Jontes, 1992; Nogales et al., 1995b). Spacings around 4.8 Å are prominent, running along the protofilament direction, along with spacings around 10 Å at about 45° to the protofilaments. Especially at low resolution, intensities on odd rows of spots perpendicular to the protofilaments are weak, reflecting the similarity of the secondary structure in the a and b monomers. At higher resolution, differences in the amino acid sequences become more important in the diffraction intensities, and the odd rows are substantially stronger. In vertical section B, the 4.8-Å spacings, corresponding mainly to beta sheet interstrand spacings, are seen to be confined to a region near the central plane. The 10-Å spacings, interpreted as
FIG. 2. Actin region corresponding to helices A (a and b) and B (c and d) (see Fig. 1) shown at different resolutions (sampling of 1 Å/pixel). The backbone of the helices is shown in yellow. The electron density at 2.1 Å, calculated with the full data, is shown in brown. The white surface in (a) and (c) shows the effect of a 6.5-Å resolution cutoff, a cone of missing data, and loss of signal parallel to the c-axis. The effect of a 4-Å cutoff is shown in (b) and (d) in blue. The c-axis runs parallel to the red lines. FIG. 3. As Fig. 2 for beta sheets A (a and b) and B (c and d) (see Fig. 1). The isosurface for the 6.5-Å simulation (white surfaces) is much lower than that used in Fig. 2. FIG. 5. Three-dimensional reconstructions for a- and b tubulin at 4 Å (a) and 6.5 Å (b) (sampling of 1 Å/pixel). The bottom monomer is assigned to be the b subunit based on the presence of an extra density identified in a previous study as the taxol binding site (T) (Nogales et al., 1995a), which in turn has been shown to be in b tubulin (Rao et al., 1992; Combeau et al., 1994). The side of the molecules facing the viewer is presumed to correspond to the inside of the protofilament in the microtubule based on comparisons of our zinc-sheet reconstructions with lower resolution helical reconstructions of microtubules (Wolf et al., 1996). Microtubule reconstructions show a smooth ridge on the outside of the protofilament and a bumpy inside surface (Kikkawa et al., 1995). The red and blue lines on (b) indicate the approximate position of the sections shown in Figs. 6a and 6b. Elements identified in those sections have here been labeled to help the reader position them in the context of the whole molecule.
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FIG. 6. Different sections through the tubulin dimer model at 4 Å resolution. Blue and pink correspond approximately to a and b monomers, respectively. The translucent white surface shows the model at 6.5 Å. Color code of axes: green runs along protofilaments, red runs through the protofilament inside out (thus, it is the direction of the c-axis or missing cone), and blue runs across from one protofilament to another. The three sections were cut parallel to these axes. (a) Section cut near the region of contact between protofilaments, on the ‘‘taxol side’’ (see red line in Fig. 5b). This section includes three clear helical regions that are present in both a and b tubulin. They have been arbitrarily labeled H1, H2, and H3. The putative taxol site is labeled T. (b) Section parallel and contiguous to that in (a) showing what looks like a five-strand beta sheet (B1; see blue line in Fig. 5b). The individual strands have been arbitrarily labeled B1,1 to B1,5. The section also includes part of two helices, H4 and H5. The 6.5-Å surface is displayed at a much lower isosurface than in (a) and (c) in order to see density corresponding to the beta strands (see also the actin models above). (c) Section corresponding to part of the outside surface of the protofilament. The first three strands of B1 can be seen end-on, B1,3 connecting to H5. Two more long helices can be seen. H6 lies at the surface and crosses over a more internal helix, H7 (see also (d)). (d) Stereo view through the center of the b subunit showing beta sheet B1 and adjacent sheet, B2, running at an angle. The figure also shows an end-on view of H1, a tilted view of H2, and a side view of H3. Another short helix, labeled H8, is also shown end-on. The section includes part of the a subunit at the bottom, i.e., the corresponding H1. 123
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TABLE I Statistics for Image Data Set Resolution range (Å)
Number of reflections
Phase residual
100–14 14–7 7–5 5–4
3121 11 477 8594 7184
28.0° 19.8° 30.0° 36.7°
spacings between helices or sheets, increase in strength away from the plane. This is seen also in section C, which cuts parallel to the protofilaments. The distribution of diffraction intensities implies that the beta strands will be constrained to orientations nearly perpendicular to the crystal plane, with beta sheets mainly perpendicular to the protofilament axis. There is no clear indication of preferred orientation of a helices, although we may expect a tendency for features running in the directions of the diagonals of the unit cell. Figure 5a is an overall view of the 4-Å map. To help in orienting the new map with respect to those previously published, Fig. 5b shows a map built from the same data set but with a resolution cutoff at 6.5 Å. We note first that all of the major features of the new 6.5-Å map were already seen in the first map (Nogales et al., 1995a), although there is an improved sense of connectivity, with longer tube-like regions lining much of the outside surface (away from the viewer). The impression of the monomer outline has become more distinct. As displayed in Fig. 5, the 4-Å map is dramatically different from the 6.5-Å map, appearing at first as a collection of scattered, disconnected densities. Upon comparison with the lower resolution map, and more careful inspection, the relation between these densities and the anticipated secondary structure becomes clear throughout much of the map. The 6.5-Å map shows many cylindrical features that are highly suggestive of a helices. Several of these had been tentatively identified earlier, but the clarity in the present map is distinctly better. On the other hand, some of the regions previously interpreted as beta sheet are nearly invisible now at 6.5 Å at the isosurface shown. As seen in the actin simulations, the density in sheet regions is lower than in helices, so that a lower contour level must be used to see them. Beta sheets then appear as flat slabs (see Fig. 6b). The situation is quite different in the 4-Å map. Here most of the apparent helices can be seen as corkscrews with a clear righthand twist. The beta strands are particularly well defined, along with side chains sitting above and below the sheets. At this resolution, even some of the loop regions show up fairly distinctly.
Figure 6 shows different sections through the tubulin dimer2 reconstruction at 4 and 6.5 Å resolution, with main secondary structure elements labeled as helices (H) or beta strands (B). The three helices in Fig. 6a, H1, H2, and H3, can be very clearly seen for both a and b tubulins. At 6.5 Å they appear as smooth cylindrical surfaces, while at 4 Å the turns can be distinguished as well as some of the bulkier residues (e.g., see H1 in the b subunit). The appearance of the three helices at 4 Å is quite different due to their different orientations with respect to the c-axis (i.e., missing cone). While H1 is almost parallel to the c-axis, and can be seen end-on in the front view of Fig. 5, H3 is almost perpendicular to it and is shown in Fig. 5 approximately in a side view (see Fig. 6d for a clearer side view of H3). Helices perpendicular to the c-axis tend to appear as spring-like densities (see also H4, H5, H6, and H7), and those running parallel (H1 and H8) have a more compact appearance. The section shown in Fig. 6b corresponds to a beta sheet region (B1). The 6.5-Å white surface has been displayed at a much lower isosurface than in Figs. 6a and 6c in order to see density corresponding to the beta strands and appears as a discontinuous slab of density. Notice that the isosurface displayed in Fig. 5b lacks flat slabs, although helical regions are well represented. At 4 Å, however, individual strands are clearly visible. This is in good agreement with what we see in the actin simulations in the previous section. Individual strands are arbitrarily labeled in the figure. At its widest point (B1,3) the sheet runs through almost the whole depth of the molecule. Bulkier residues in the strands can be distinguished sticking toward and away from the viewer. The section also includes part of two helices that run around the sheet, perpendicular to the strands: H4, on the inside microtubule surface (front in Fig. 5), and H5, toward the outside surface. There is a very clear connection between B1,3 and H5, especially in the a subunit (see also Fig. 6c). A section corresponding to part of the putative outside surface of the protofilament (smoother surface, away from the viewer in Fig. 5) is displayed in Fig. 6c. This section shows the first three strands of B1 end-on, with B1,3 connecting to H5. There are two more helices, H6, which lies on the outside surface of the protofilament and crosses over a more internal helix, H7. Finally, Fig. 6d shows a stereo view through the center of the b-tubulin monomer
2 The definition of the dimer is still a matter of controversy. Given the similarity between the a–b and b–a longitudinal interactions it is difficult to assert which is the principal contact. We leave this point for further work.
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FIG. 4. Three-dimensional electron diffraction intensity data. Intensities are shown on three planes that cut through the data in three dimensions. Plane A is parallel to the plane of the crystal, with the protofilaments parallel to the direction marked h. Continuous intensities are seen along the reciprocal lattice lines in planes B and C.
showing beta sheet B1 and an adjacent sheet, B2, running at an angle. The figure also shows an end-on view of H1, a tilted view of H2, and a side view of H3, while H7 has been cut right through the middle. Another short helix, labeled H8, is also shown endon. The several large protrusions in this helix are most likely aromatic residues. The handedness of the reconstruction had been inferred previously by the assignment of the faces corresponding to inside and outside of the microtubule and by comparison with earlier low-resolution reconstructions (Wolf et al., 1996). At 4 Å resolution, the helices in this reconstruction are all righthanded (e.g., Fig. 6c), as is the twist of the beta
sheets (Fig. 6d), thus confirming that the hand has been presented correctly. In the 4-Å map, differences between the two monomers are dominated by densities that are presumably related to side chains and larger regions in the contact areas between monomers. There is an area of extra density in a tubulin at one end of helix H2 that is involved in the main lateral contact between protofilaments in the sheets. There is also an extra loop (or more ordered loop) visible in one of the longitudinal contacts, although it is difficult to decide whether it belongs to the a or b monomer. The putative taxol site (T in Figs. 5 and 6d) is the most prominent density in the b monomer that is not
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present in a. There is almost enough of a distinctive shape to this density to allow modeling of the orientation of the taxol molecule. DISCUSSION
Electron crystallography has been a fruitful tool for the study of tubulin structure, especially in the absence of high-resolution X-ray data. The methodology of electron crystallography has developed over the recent years to the point that it is possible to construct accurate atomic models of proteins from electron microscope data, but the progress in this work is still substantially slower than for an X-ray structure determination. The atomic model of bacteriorhodopsin (bR) (Henderson et al., 1990), the first determined by electron crystallography, required 15 years from the time that the first low-resolution structure was published, mainly because much of the technology had to be developed in the process. The light-harvesting complex (LHC) (Kuhlbrandt et al., 1994) was solved just 5 years after the first high-resolution projections were obtained, using the techniques that had been put in place by the previous work. We had expected that the tubulin structure would proceed even faster, given the advances in methodology in the last few years. However, the structure of tubulin is far more complex than that of bR. The basic 3-D structure of bR, seven transmembrane helices, was clear from the low-resolution map. The orientation of the helices, roughly perpendicular to the crystal plane, made it particularly easy to identify them with limited resolution. Only about a third of the tubulin structure is expected to be helix, and there is apparently no preferred direction with respect to the plane of the sheet. Extending the resolution of the bR map from 7 to 3.5 Å, even with a very anisotropic point spread function, allowed identification of sufficient structure within the helices to assign side chains to densities in the map. Most of the data for the bR structure were obtained from specimens tilted up to 45°, and the number of images was only about two-thirds of what we have in the tubulin data. Using electron diffraction amplitudes from specimens tilted up to 60°, the bR map has been substantially improved (Grigorieff et al., 1996). More of the loop regions are resolved, although the atomic model has very high temperature factors in nonhelical regions. The light-harvesting complex, on the other hand, contains a significant amount of loop within the molecule. Using a set of 79 images taken at tilts up to 60° the loop structure was better resolved than that in bR. Part of the improvement may have been a result of better sampling of the LHC data, due to the higher symmetry of the structure. Having P321
symmetry, the LHC has six monomers in the unit cell, compared to three for bR. Correspondingly, the zinc sheets have two tubulin heterodimers per unit cell, which has the effect of increasing the number of images required for the complete sampling of the data. The current data set for tubulin appears to be substantially more complete, and extends to higher angles, than that originally used for bR. The point spread function in our map is thus significantly more isotropic than in the bR map. This improvement in the data is reflected in the appearance of noticeably more detail, even in nonhelical regions, than in the bR map. The amount of detail that is already clearly visible in the present map is sufficient to allow us to begin the identification of specific secondary structure regions. Much of the molecule can now be seen in the 4-Å map to be composed of identifiable secondary structure. From secondary structure predictions and spectroscopic analyses we can expect on the order of 25% of the structure to be in sheet, 35% in helix, and the other 40% in loops, turns, and other structure (de Pereda et al., 1996). Regions identified to date would account for roughly 75% of the expected sheet and 90% of the helix. The secondary structure interpretation is now complete enough to allow us to begin searching protein structure databases to identify possible folding motifs within the tubulin structure. There are clear indications of densities for side chains in both helix and sheet regions in the map, and it should soon be possible to begin fitting the peptide chain to the map on the basis of the distribution of bulky side chains. Modeling some of the regions of the sequence that are strongly predicted to be helical should provide a firm start for the atomic model. Fitting the peptide will be helped by the specific similarities and differences between the a and b sequences and by the constraints of a growing body of biochemical, biophysical, and genetic data (Luduen˜a et al., 1992; Burns and Surridge, 1993; de Pereda et al., 1996). However, as expected at this resolution, the path of the backbone through loops connecting beta strands and a helices is not yet completely clear. Further extension of the resolution to 3.5 Å and addition of more data to improve sampling should resolve most of these unclear regions. REFERENCES Baumeister, W., Barth, M., Hegerl, R., Guckenberger, R., Hahn, M., and Saxton, W. O. (1986) Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans, J. Mol. Biol. 187, 241–253. Baumeister, W., and Typke, D. (1993) Electron crystallography of
TUBULIN SECONDARY STRUCTURE proteins: State of the art and strategies for the future, MSA Bull. 23, 11–19. Burns, R. G., and Surridge, C. D. (1993) Tubulin: Conservation and structure, in Hyams, J. S., and Lloyd, C. W. (Eds.), Microtubules pp. 3, 32 Wiley, New York. Collaborative Computational Project, N. 4. (1994) The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallogr. D50, 760–763. Combeau, C., Commerc¸on, A., Mioskowski, C., Rousseau, B., Aubert, F., and Goeldner, M. (1994) Predominant labeling of bover a-tubulin from porcine brain by a photoactivatable taxoid derivative, Biochemistry 33, 6676–6683. de Pereda, J. M., Leynadier, D., Evangelio, J. A., Chacon, P., and Andreu, J. M. (1996) Tubulin secondary structure analysis, limited proteolysis sites and homology to FtsZ, Biochemistry 35, 14203–14215. Downing, K. H., and Jones, J. (1992) Projection map of tubulin in zinc-induced sheets at 4 Å resolution, J. Struct. Biol. 109, 152–159. Glaeser, R. M., Tong, L., and Kim, S. H. (1989) Three-dimensional reconstructions from incomplete data: Interpretability of density maps at ‘‘atomic’’ resolution, Ultramicroscopy 27, 307–318. Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M., and Henderson, R. (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin, J. Mol. Biol. 259, 393–421. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman,
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E., and Downing, K. H. (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy, J. Mol. Biol. 213, 899–929. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Atomic structure of the actin:DNase I complex, Nature 347, 37–44. Kikkawa, M., Ishikawa, T., Wakabayashi, T., and Hirokawa, N. (1995) Three-dimensional structure of the kinesin headmicrotubule complex, Nature 376, 274–277. Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Atomic model of plant light-harvesting complex by electron crystallography, Nature 367, 614–621. Luduen˜a, R. F., Banerjee, A., and Khan, I. A. (1992) Tubulin structure and biochemistry, Curr. Opin. Cell Biol. 4, 53–57. Nogales, E., Wolf, S. G., Khan, I. A., Luduena, R. F., and Downing, K. H. (1995a) Structure of tubulin at 6.5 Å and location of the taxol-binding site, Nature 375, 424–427. Nogales, E., Wolf, S. G., Zhang, S. X., and Downing, K. H. (1995b) Preservation of 2-D crystals of tubulin for electron crystallography, J. Struct. Biol. 115, 199–208. Rao, S., Horwitz, S. B., and Ringel, I. (1992) Direct photoaffinity labeling of tubulin with taxol, J. Natl. Cancer Inst. 84, 785–788. Wolf, S. G., Nogales, E., Kikkawa, M., Gratzinger, D., Hirokawa, N., and Downing, K. H. (1996) Interpreting a mediumresolution model of tubulin: comparison of zinc-sheet and microtubule structure, J. Mol. Biol. 263, 485–501.