A Major Conformational Change in p97 AAA ATPase upon ATP Binding

Molecular Cell, Vol. 6, 1485–1490, December, 2000, Copyright 2000 by Cell Press

A Major Conformational Change in p97 AAA ATPase upon ATP Binding Isabelle Rouiller,* Virginia M. Butel,† Martin Latterich,†§ Ronald A. Milligan,* and Elizabeth M. Wilson-Kubalek*‡ * Department of Cell Biology Scripps Research Institute La Jolla, California 92037 † Salk Institute La Jolla, California 92037

Summary AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners. Introduction Virtually all major cellular pathways involve at least one member of the AAA ATPase family or the closely related AAA⫹ ATPase family (Patel and Latterich, 1998; Vale, 2000). These AAA/AAA⫹ ATPases (ATPases associated with different cellular activities) participate in assembly, operation, and disassembly of diverse protein and nucleoprotein machines. They share an evolutionarily conserved ⵑ250 amino acid sequence predicted to form a common structural core (Neuwald et al., 1999). AAA ATPases contain one (type I) or two (type II) of these motifs per monomer. Despite their critical role in many cellular pathways and their conserved structural motif, their mode of action is not well understood. In particular, the link between the ATPase catalytic cycle and the conformational changes leading to their biological activity has yet to be elucidated. This study focuses on one representative AAA protein, p97, the mammalian ortholog of yeast Cdc48p and archaeon VAT. This protein is central to organelle assembly in dividing cells and functions in the homotypic membrane fusion process of early secretory organelles, such as the endoplasmic reticulum and the Golgi apparatus (Patel and Latterich, 1998). The homohexamer p97 has been shown to interact with syntaxin V, a member of the t-SNARE family, via the adaptor protein p47 (Kondo ‡ To whom correspondence should be addressed (e-mail: kubalek@

scripps.edu). § Present address: Diversa Corporation, 10665 Sorrento Valley Road, San Diego, California 92121.

et al., 1997; Rabouille et al., 1998; Roy et al., 2000). Current models suggest that p97, in conjunction with its adaptor protein p47, functions in disassembling t-tSNARE complexes in a manner similar to the related N-ethylmaleimide-sensitive fusion protein (NSF) and its adaptor protein ␣-SNAP with v-t-SNARE complexes (Clary and Rothman, 1990; Hohl et al., 1998; Patel et al., 1998; Roy et al., 2000). NSF and p97 have been proposed to use the energy provided by ATP hydrolysis to disassemble SNARE complexes and prepare the components, t- and/or v-SNARE, for another round of membrane fusion (So¨llner et al., 1993; Hanson et al., 1997). However, many lines of evidence suggest that NSF and p97 carry out different functions in the membrane fusion process. First, if both proteins prime the same SNAREs for subsequent fusion events, the need for two distinct ATPases is not clear. The same SNAREs that function in vesicle fusion also function in organelle membrane fusion (Patel et al., 1998; Rabouille et al., 1998). Second, p97 can be found complexed with its adaptor, p47, even when not bound to a membrane (Kondo et al., 1997). On the other hand, NSF’s adaptor, ␣-SNAP, has to first bind to a SNARE complex before recruiting NSF from the cytosol (Wilson et al., 1992). Last, when complexed with p47, p97 has a lower rate of ATP hydrolysis (Meyer et al., 1998), whereas NSF ATPase activity is stimulated by ␣-SNAP (Morgan et al., 1994). Taken together, these lines of evidence suggest that the superficially very similar NSF and p97 ATPases have different molecular functions in membrane fusion mediated by their structurally unrelated adaptors. Additionally, the p97/Cdc48p ATPase has also been shown to interact with Ufd1p and Npl4, factors required for ubiquitin-dependent protein degradation and nuclear transport, respectively (Meyer et al., 2000), and with a DNA unwinding factor that functions in DNA replication (Yamada et al., 2000). These interactions do not require the adaptor protein p47. These data suggest that p97 is involved in a variety of cellular pathways and that the specificity of its function is dependent on its binding partners (Patel and Latterich, 1998). To investigate how p97 uses ATP hydrolysis to induce a conformational change in its binding partners, we used single-particle analysis to investigate the structural differences in p97 in various nucleotide states. For this purpose, we looked at the conformation of p97 in a nucleotide-free state and in the presence of AMP–PNP and ADP. We also studied the interaction of p97 with one of its adaptor proteins, p47, to understand how structural changes in p97 may be translated to an associated organelle or SNARE complex. Results Homogeneous preparations of native bovine p97 (Figure 1A) were studied by cryo-EM and image processing. The majority of the p97 complexes consistently adopted a preferred en face orientation on the EM grids (Figure 1B). We calculated projection maps of these top views of

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perimeter and a well-defined handedness. A simple explanation for the observed changes is that a large portion of each monomer rotates upon binding ATP (arrows in Figure 2E). In support of this rotation model is the observation that the angle between the center–p2 axis and p2–p3 axis decreases by 11⬚ on nucleotide binding (58⬚–47⬚; Figures 2D and 2E).

Figure 1. Purification and Imaging of p97 (A) Coomassie blue–stained SDS-PAGE gel of native p97 purified from bovine liver and murine p47 purified as a recombinant protein from E. coli. (B) Cryoelectron micrograph showing a field of p97 complexes purified in the absence of nucleotide. Scale bar, 200 A˚.

p97 under different nucleotide conditions. Preliminary maps were first calculated without applying symmetry and clearly showed a 6-fold symmetric complex. The symmetry was also carefully verified by rotational power spectra analysis. We then applied 6-fold symmetry to the data set and calculated new projection maps (Figures 2A–2F), whose resolutions were estimated to be ⵑ20–25 A˚. Nucleotide-Free p97 We first calculated the projection map of p97 that had been treated with apyrase to remove nucleotide (Figures 2A and 2D). Almost identical maps were obtained with p97 that had been prepared in the absence of nucleotide. Under these nucleotide-free conditions, p97 is a hexameric complex. The protein monomers are arranged around a central hole of ⵑ25 A˚ diameter and extend outwards to a radius of ⵑ84 A˚. Adjacent to the central hole are six regions of low density (l in Figure 2D). These low-density regions are defined by a ring of six alternating inward-pointing peaks (p1) and six outward-pointing peaks of density (p2). The peaks p1 and p2 are of roughly equal size. At a radius of ⵑ78 A˚ there is a small, weak, but consistently observed density (p3) connected to p2. Its asymmetric location on one side of p2 confers a weak handedness to this view of the entire hexamer. Conformational Change upon Binding AMP–PNP To investigate the ATP-bound structure of the complex, we added a ⵑ1000-fold molar excess of a nonhydrolyzable ATP analog, AMP–PNP, to p97 that had been purified in the absence of nucleotide. Parallel experiments using radiolabeled nucleotide showed that ⵑ95% of p97 can bind nucleotide under conditions similar to those used for cryo-EM (data not shown). The projection map of p97 with AMP–PNP (Figures 2B and 2E) is dramatically different from the map of nucleotide-free p97. Although the central hole and adjacent low-density regions (l) of the complex seem to be unchanged, there is considerable rearrangement of the density peaks in the ring. Densities attributed to p1 peaks are weaker than in the nucleotide-free condition. The p2 peaks, as well as being more massive, are now at a slightly higher radius (an increase of ⵑ5 A˚) and are more asymmetric. Overall, the complex now has a much more scalloped outer

p97–ADP Complex To visualize the conformation of p97 at the end of the ATPase cycle, we incubated nucleotide-free p97 with ADP, imaged the resulting complex by cryo-EM, and calculated an average projection map. The projection map (Figures 2C and 2F) is dramatically different from the map obtained with the nucleotide-free p97 (demonstrating that ADP is bound) and is very similar to the map of p97–AMP–PNP. A detailed comparison of the AMP–PNP and ADP maps revealed a small but statistically significant difference at the edge of the low-density regions adjacent to p1 (circled in Figure 2F). This finding suggests that there is a relatively small localized rearrangement of the structure between the ATP and ADP states. Modeling The p97 monomer is composed of three domains of similar molecular weight: an N-terminal domain followed by two similar, but not identical, AAA domains (called D1 and D2). Despite sequence variation, X-ray crystal structures of the three divergent AAA domains are extremely similar (Guenther et al., 1997; Lenzen et al., 1998; Yu et al., 1998; Bochtler et al., 2000), suggesting that the overall fold is conserved in all AAA domains. Additionally, since p97 and NSF are both type II AAA ATPases involved in membrane fusion, we reasoned that the NSF D2 crystal structure (solved in the presence of ATP; Lenzen et al., 1998; Yu et al., 1998) represents a good model for both the D1 and D2 domains of p97. To help interpret our results, we calculated a projection map of NSF D2 hexamer and then filtered it to exclude structural details finer than 20 A˚ resolution (Figures 2G and 2I). The good correspondence between features in this model and the EM data of p97 is striking and indicates that the D1 and D2 domains of p97 sit on top of each other. We used two stacked NSF D2 hexamers (representing D1 and D2 of p97) rotated one against the other and calculated projection maps at stepwise increments. This experiment demonstrated that the hexamers cannot be more than 20⬚ out of register (data not shown). Given the modeling results described above, it is reasonable to interpret the p97 projection maps using the NSF D2 crystal structure. The N subdomains of NSF D2 (Figure 2H1), consisting of parallel ␤ sheets surrounded by ␣ helices and a nucleotide binding pocket, are located in the center, and the four-helix bundle C subdomains are located at the periphery of the hexamer (Figure 2H2). Based on this observation, we argue that in our projection maps of p97, the low-density peaks (l) around the central hole and the p1 peaks must comprise the N subdomains of the p97 AAA domains. The p2 peaks at the periphery of the p97 projection maps must therefore include the C subdomains. Furthermore, the

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Figure 2. Conformations of p97 and Comparison with NSF D2 Six-fold symmetrized average projection maps of p97 complexes, in the absence of nucleotide (A), bound to AMP–PNP (B), and to ADP (C). Gray scale maps show the protein densities as white on a dark background. Contour plots of the nucleotide-free p97 (D), p97–AMP–PNP (E), and p97–ADP (F) emphasize the main features: a central hole, six regions of low density (l), surrounded by six inward-pointing peaks (p1), six outward-pointing peaks (p2), and six small peaks (p3) at the periphery. Upon binding of nucleotide, the domain encircled in (D) rotates (arrows in E). One of the six areas, which contains the small but statistical difference between the AMP–PNP and ADP state, is circled in (F). The NSF D2 domains from the PDF files of NSF D2 (Protein Data Bank ID code 1NSF; Yu et al., 1998) were imported into Spider, converted into densities, and low-pass filtered at 20 A˚ resolution. The en face projection was calculated, and displayed as a gray scale map (G) and contour map (I). The calculated projection map formed by six NSF D2 domains was split in two halves: (H1) showing only the N subdomains and (H2) only the C subdomains. Scale bar, 40 A˚.

p2 peaks of the p97 maps are much larger than the corresponding densities in the NSF D2 calculated map and confer a strong handedness to the projection maps, which strongly suggests that the N-terminal domain of p97 is responsible for this difference. However, these additional densities seem to be too weak to account for all of the 30 kDa of the N-terminal domains, suggesting that these N-terminal domains are flexible. p97–p47 ATP Complex In the first part of this study, we have established that ATP binding to the p97 complex induces a large conformational change in p97. To study how this conformational change could be transmitted to its binding partners and in particular to the membrane fusion complex, we observed p97 bound with its adaptor protein p47 by cryo-EM in the presence and absence of nucleotide. As with p97 alone, the p97–p47 complexes preferentially adopted an en face orientation on the grids under all experimental conditions. These top views of p97–p47 were used for structural analysis. Similar to the p97 analysis, we calculated the projection map of the p97– p47 complexes without applying symmetry. The average clearly showed a 6-fold symmetric molecule, which was confirmed by using rotational power spectrum analysis. Figures 3A and 3B show a 6-fold symmetrized average of p97 in the presence of ATP at ⵑ20–25 A˚ resolution. Maps obtained in the presence of AMP–PNP were virtually identical (data not shown). Six additional densities are clearly visible around the

periphery of p97, which strongly suggests that six p47 molecules bind to the p97 hexamer. A careful comparison of the p97 and p97–p47–ATP maps allowed us to conclude that when bound to p47 and ATP, p97 remains in a state similar to when bound to ATP alone. To further analyze the difference seen between the p97–AMP–PNP and p97–p47–ATP/AMP–PNP maps, we calculated a difference map between these complexes. The difference map showed 12 peaks of additional mass, one on each side of the small densities previously attributed to the flexible N domain of p97 (contoured in Figure 3A). The total density corresponding to the p97–p47–ATP/AMP–PNP averages was approximately twice the density of p97 alone. This increase of apparent mass was calculated by summing up the pixel values associated with the proteins. The analysis, although approximate, suggests that this difference is greater than the expected mass of six added p47 monomers. As noted earlier, the N-terminal domains of p97 (ⵑ30 kDa each) are flexible, and their mass is consequently underrepresented in the average projection map of p97 alone. We infer that, when bound to p47, the N-terminal domains of p97 become locked into a more rigid position and thereby contribute to the large density difference observed. Nucleotide-Free and ADP p97–p47 Complexes Analysis of both the nucleotide-free and ADP p97–p47 complexes gave similar results. In both cases, global averages showed a significant deviation from any rota-

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Figure 3. Conformations of the p97–p47 Complex The 6-fold symmetrized average projection map of the p97–p47 complex in the presence of ATP was displayed as a gray scale map (A) and a contour map (B). This average has been aligned with the average of p97–AMP– PNP (Figure 2B). The areas of weak densities (l) and the peaks of density p1 and p2 are indicated. Six additional areas of density at the periphery of the complex are clearly visible. The difference map between p97–p47– ATP and p97–AMP–PNP was calculated, and a contour map representing one sixth of the extra mass is shown on projection map A. In the absence of nucleotide, the average projection map of the p97–p47 complex is less ordered, and the densities attributed to p47 are not well defined and appear to be less rigidly bound. Four independent averages of the p97–p47 (nucleotide-free) complexes are shown in (C). For comparison, four similarly obtained averages of p97–p47–ATP are shown in (D). Scale bar, 40 A˚.

tional symmetry. Examples of independent averages of the nucleotide free are shown in Figure 3C. A similar set of averages in the ADP state (data not shown) is indistinguishable. For comparison, the p97–p47–ATP images were analyzed in a similar manner. The two data sets reveal that whereas the p97–p47–ATP averages (Figure 3D) clearly show six p47 monomers regularly spaced at the periphery of p97 hexamer (averages almost identical to Figure 3A), the ADP and nucleotidefree structures are highly variable (Figure 3C). Some averages show fewer than six p47 monomers, and the positions of p47 vary with respect to p97. These observations suggest that in the ADP and nucleotide-free conditions, the p47 molecules bind more weakly to p97 and are rather mobile.

lished lower resolution EM studies (Peters et al., 1992). Our studies are also in agreement with the study of the p97 ortholog VAT (Rockel et al., 1999), which showed that the N-terminal domains of VAT protruded from the periphery of the molecule and were mobile. In the VAT complex, the two AAA domains were arranged on top of each other with a small stagger. Modeling experiments we carried out with NSF D2 also ruled out a rotational stagger greater than 20⬚ in the p97 complex. However, our analysis would be unable to detect a small rotation of the two AAA domains with respect to each other during the nucleotide cycle.

Discussion In the first part of the study, we observed p97 by cryo-EM and calculated projection maps of this complex under different nucleotide conditions. The p97 monomer contains two AAA domains (called D1 and D2) and an N-terminal domain. Since the structure and function of p97 and NSF are expected to be similar, we used a calculated projection map of NSF D2 to interpret the p97 projection maps. Our results are compatible with a model for p97 in which the D1 and D2 domains lie on top of each other with a mobile N-terminal domain at the periphery of the complex. D1 and D2 seem to have the same overall organization as NSF D2 with their N subdomains at the center of the hexamer surrounded by their C subdomains. The detailed architecture of p97 shown here is also in agreement with previously pub-

Figure 4. Model for p97–p47 in Action Model for the p97–p47 function in disassembling an organelle (t-tSNARE complex or a SNARE inhibitor–t-SNARE complex). In the absence of nucleotide, the p47 molecules (shadowed in gray) are weakly bound to the periphery of the p97 hexamer and are mobile. This freedom of movement allows them to bind to the organelle complex (schematically represented here as two intertwined structure elements). Upon ATP binding, the p47 molecules become rigidly attached at the periphery of the p97 hexamer. This change from a disordered to an ordered complex could serve to pull the organelle apart.

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We observed a major conformational change between the nucleotide-free and ATP states and a comparatively minor difference between the ATP and ADP states. We have not examined the p97–ADP–Pi state and can therefore not rule out the possibility of a conformational change at this step of the ATP hydrolysis cycle. However, a separate study showed that a mutant Cdc48p (the p97 ortholog in yeast) was fully functional in cell cycle progression and membrane fusion when ATP hydrolysis was abolished (N. Olivieri, N. Levine, and M. L., unpublished data). Together these observations suggest that the structural change during nucleotide binding is the only one needed for the relevant biological activity. In this model, ATP hydrolysis would accelerate nucleotide release, regenerating nucleotide-free p97 and preparing it for another activity cycle; that is, ATP hydrolysis would speed up the cycle. This mechanism is reminiscent of the action of kinesins where nucleotide binding rather than hydrolysis drives the power stroke (Vale and Milligan, 2000). Surprisingly, despite the sequence conservation between NSF and p97 and the expected conserved fold of their domains, the two molecules behave quite differently during the ATPase cycle. Consequently, our model for p97 complex is very different from those suggested for NSF. In the case of NSF, ATP hydrolysis is essential for the disassembly of SNARE complexes (Whiteheart et al., 1994). EM studies have shown that NSF forms a narrow cylinder in the presence of nucleotide and dissociates in the absence of nucleotide. The N-terminal domains undergo a large conformational change between the ATP and ADP states (Hanson et al., 1997). Another model, based on the crystallization of the N-terminal domain of NSF as a trimer, proposed that three of the six N-terminal domains point inwards and three outwards in both the ATP and ADP states and interchange upon ATP hydrolysis (May et al., 1999). It has also been argued that the crystallization of NSF as a trimer was a crystallographic artifact (Yu et al., 1999). In contrast, our data show that p97 is a symmetric hexamer in all the nucleotide states examined and that it is nucleotide binding, not hydrolysis, that drives the major conformational change. In the second part of the study, we analyzed the interaction of p97 with p47, its adaptor protein, under different nucleotide conditions. Previous studies have suggested that three p47 molecules bind at the center of the p97 hexamer (Kondo et al., 1997; Rabouille et al., 1998). Our cryo-EM studies directly visualized six p47 molecules bound to the periphery of the p97 complex in the presence of ATP, supporting the observation that the inhibition of p97 ATPase activity by p47 is greatest when there is an equimolar ratio of the two proteins (Meyer et al., 1998). In nucleotide-free and ADP states, the p47 molecules are more weakly bound and more flexibly attached to the p97 hexamer. We propose that in these states, this flexibility of attachment allows the p47 adaptor molecules to move around and bind more easily to other cofactors, such as the SNARE complex or a SNARE inhibitor. When ATP binds p97, the p47 adaptors become rigidly attached at the periphery of the hexamer in a splayed manner. This change from a more mobile to

a rigidly locked position of p47 could apply a stretching force to bound components to pull them apart (Figure 4). The striking observation that ATP binding, not ATP hydrolysis, leads to a major conformational change in the p97 molecule reveals a new paradigm for how p97 functions in membrane fusion events and other cellular mechanisms, such as protein turnover. This modus operandi is unusual for molecular machines involved in assembly and disassembly of protein complexes. These machines, such as in the chaperone GroEl and the proteasome, usually couple ATP hydrolysis to protein-folding reactions. Since p97 and its orthologs have been hypothesized to act as protein unwindases similar to the structurally related DNA helicases, this either hints at a novel protein-unfolding mechanism or suggests that this interesting ATPase can couple ATP/ADP exchange to molecular disassembly of large protein complexes. By attaching to protein components and temporarily or permanently prying them apart, p97–p47 could allow other proteins to exit or enter the complex. Numerous proteins belong to this family of ATPases. Given the high degree of sequence conservation within the AAA domain, it is likely that other AAA molecules link ATP binding to structural changes, in contrast to previous hypotheses. Employing similar structural methods to study other members of the AAA and AAA⫹ families will shed light on how the nucleotide cycle links structure to biological function. Experimental Procedures Purification of p97 and p47, and Preparation of Frozen Specimen p97 was purified from bovine liver in the absence of nucleotide as will be reported elsewhere. Murine p47 was purified as a recombinant protein from E. coli. In membrane fusion assays, this bovine p97– murine p47 complex is indistinguishable in specific biological activity from all bovine or all mouse complexes (unpublished observations). To study the conformational change during the ATPase cycle, p97 was incubated for 30 min before freezing in the presence of 2 mM ADP, ATP, or AMP–PNP, in buffer (40 mM HEPES, 200 mM NaCl, and 2 mM MgCl2 [pH 7.5]). To remove residual nucleotides, p97 was treated with apyrase (10 units/ml; Sigma). Experimental results show that the projection map of p97 in the purification buffer was identical to the projection map of p97 treated with apyrase. For the study of the p97–p47 complex, p97 was incubated with excess p47 (molar ratio of 1:3) in the above buffer. Samples were incubated with 2 mM nucleotide for 30 min or were treated with apyrase before freezing. Experimental results were identical whether nucleotide was added to p97 before or after incubation with p47. Electron Microscopy Frozen hydrated grids of p97 or p97–p47 complexes were observed by a Philips CM200FEG microscope at 120 KV. Electron micrographs were recorded under low-dose conditions at a nominal magnification of 50,000⫻ and at an underfocus of ⵑ1.8 ␮m. The quality of micrographs was assessed by optical diffraction. Image Analysis Micrographs were digitized on a Zeiss scanner with a 7 ␮m pixel size. Groups of 3 ⫻ 3 pixels were averaged, giving a pixel size of 4.2 A˚ on the specimen. Images were analyzed, unless otherwise indicated, using the SPIDER package of image analysis programs (Frank et al., 1996). For each condition, a minimum of 2000 particle images in the en face orientation was selected. Normalized images were first aligned using reference-free alignment. This step was followed by classification to check homogeneity and discard bad images. The average obtained was used to determine the top or bottom orientation of

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the particles and refine the alignment using a reference-based alignment subroutine repeated five times. Under all conditions, averages were first calculated without imposing symmetry. When 6-fold symmetry was apparent for the initial averages, the symmetry was further confirmed by statistical analysis using the program Rota-stat (Kocsis et al., 1995). Finally, after testing for symmetry, the best 900 particles (based on the highest correlation coefficient to the final average) were 6-fold symmetrized, and a final average was calculated for each condition. Resolution was estimated based on the Fourier shell correlation criteria with a cut-off value at 0.5. The final averages were then filtered to the estimated 20 A˚ resolution. Student’s t tests were performed using the best 900 particles for the p97 study between the nucleotide-free state, the ATP/AMP–PNP, and the ADP state. The processing of the p97–p47 complex images was performed as described above for the p97 complex, by using reference-free and reference-based alignment procedures, without applying any symmetry. The alignment procedure was repeated by 6-fold symmetrizing the reference at each step. This was followed by correspondence analysis and a hierarchical classification, focusing on the area containing the p47 molecules. Classes containing ⵑ150 images were used to observe the variation in binding and localization of the p47 molecules. Acknowledgments This work was supported by grants GM54729 (to M. L.) and AR39155 (to R. A. M.) from the NIH, and the Joe W. and Dorothy Brown Foundation (to M. L.). M. L. is a Pew Scholar. We thank Pawel Penczek, Michael Radermacher, Brian Adair, Brian Sheehan, and Francisco Asturias for helpful discussion over computational methods, Dan LaCaze, Tammy Lai, and Robert Robinson for help with protein purification, and Senyn Choe and Melanie Richard for stimulating discussions. Received August 25, 2000; revised October 18, 2000. References Bochtler, M., Hartmann, C., Song, H.K., Bourenkov, G.P., Bartunik, H.D., and Huber, R. (2000). The structures of HsIU and the ATPdependent protease HsIU-HsIV. Nature 403, 800–805. Clary, D.O., and Rothman, J.E. (1990). Purification of three related peripheral membrane proteins needed for vesicular transport. J. Biol. Chem. 265, 10109–10117. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199. Guenther, B., Onrust, R., Sali, A., O’Donnell, M., and Kuriyan, J. (1997). Crystal structure of the ␦⬘ subunit of the clamp-loader complex of E. coli DNA polymerase III. Cell 91, 335–345.

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