The Tata Component of the Twin-Arginine Protein Transport System Forms Channel Complexes of Variable Diameter

The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter

Ulrich Gohlke*†, Lee Pullan*‡, Christopher A. McDevitt§, Ida Porcelli§, Erik de Leeuw§, Tracy Palmer¶ʈ, Helen R. Saibil*, and Ben C. Berks§

*Institute of Structural Molecular Biology, School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom; §Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; ¶School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; and ʈDepartment of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom

Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved June 8, 2005 (received for review April 29, 2005) The Tat system mediates Sec-independent transport of folded diameter (15, 17). TatBC has been shown to act as the receptor precursor proteins across the bacterial plasma membrane or the element of the translocation pathway (13, 16, 18), and TatC has chloroplast thylakoid membrane. Tat transport involves distinct been shown to contain the primary binding site for the signal high-molecular-weight TatA and TatBC complexes. Here we report peptide (18). The function of TatA is less clear. It is known that the 3D architecture of the TatA complex from Escherichia coli TatA is required subsequent to substrate recognition by the obtained by single-particle electron microscopy and random con- TatBC complex (16, 18, 19), and this has led to the suggestion ical tilt reconstruction. TatA forms ring-shaped structures of vari- that TatA constitutes the protein-conducting channel of the Tat able diameter in which the internal channels are large enough to system. Recent chemical crosslinking studies suggest that TatA accommodate known Tat substrate proteins. This morphology transiently associates with the TatBC–substrate complex during strongly supports the proposal that TatA forms the protein- active protein translocation (18, 19). conducting channel of the Tat system. One end of the channel is Like other protein translocation systems, the Tat pathway is closed by a lid that might gate access to the channel. On the basis presumed to form an aqueous transmembrane channel for of previous protease accessibility measurements, the lid is likely to protein transport. It is vital that there is no ion leakage through be located at the cytoplasmic side of the membrane. The observed this channel, because the bacterial cytoplasmic membrane has to variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of maintain transmembrane proton and other ion electrochemical the channel to the size of the substrate being transported. Such gradients to drive essential cellular functions. To preserve the dynamic close packing would provide a mechanism to maintain the ionic permeability barrier of the membrane, opening of the Tat membrane permeability barrier during transport. channel must be gated by substrate. In addition, a mechanism is required to prevent ion leakage around the translocating sub- conical tilt reconstruction ͉ electron microscopy ͉ Tat protein transport ͉ strate protein during transport. These considerations also apply three-dimensional structure ͉ twin-arginine signal peptide to the Sec channel present in the same membrane. However, the mechanistic challenges posed for the Tat channel are consider- any proteins function in extracytoplasmic compartments ably greater than for the Sec channel. Because some folded E. Msuch as the periplasm in Gram-negative bacteria or the coli Tat substrates are close to 70 Å in diameter (20), the Tat chloroplast in plants. In bacteria, most exported proteins are system must be able to form a channel of this size as opposed to moved across the cytoplasmic membrane by the Sec pathway (1). the 12-Å channel needed to thread unfolded chains by the Sec This translocation occurs by a threading mechanism in which the translocase (1). Concerted helix tilting in the membrane may be substrate adopts an extended conformation. By contrast, the Tat sufficient to gate the pore in the Sec apparatus (1), but a radically system exports folded proteins across the cytoplasmic membrane different mechanism seems necessary to provide the huge (2). Substrates of the Tat pathway have N-terminal signal conformational change required to gate the Tat channel. An sequences containing an S-R-R-x-F-L-K consensus motif in additional challenge for the Tat system is that, in contrast to the which the arginine residues are almost invariant (3). Tat trans- Sec pathway, substrate proteins vary widely in size. If the channel port is energized by the transmembrane proton electrochemical needs a diameter of 70 Å to accommodate the largest substrates, gradient (4). The Tat system is vital for many bacterial processes, how is ion leakage between the substrate protein and the walls including energy metabolism, formation of the cell envelope, of the channel prevented when the smallest substrates (20–30 Å biofilm formation, heavy metal resistance, nitrogen-fixing sym- in diameter) (21) are being transported? biosis, and bacterial pathogenesis (5). A Tat system is also found In this report we present the 3D density maps of the TatA in the thylakoid membrane of plant chloroplasts, where it plays complex from E. coli obtained by random conical tilt electron an essential role in the biogenesis of the photosynthetic electron microscopy of negatively stained specimens. This is the first 3D transport chain (6). structure determination for any component of the Tat translo- In Escherichia coli, the minimal components of the Tat cation pathway. It strongly supports the hypothesis that TatA translocation system are the integral membrane proteins TatA, forms the protein-conducting channel of the Tat system and TatB, and TatC (7–11). Attempts to purify the Tat components suggests possible mechanisms by which the channel can transport from E. coli membranes have led to the identification of two distinct high-molecular-mass complexes, one corresponding to TatA and the other containing predominantly TatB and TatC This paper was submitted directly (Track II) to the PNAS ofﬁce. (12–15). Both types contain multiple subunits per complex. †To whom correspondence should be sent at the present address: PSF Biotech AG, Analogous complexes have been described in de-energized Heubnerweg 6, 14059 Berlin, Germany. E-mail: [email protected]. thylakoids (16). Projection maps of Tat complexes obtained by ‡Present address: Department of Biochemistry and Molecular Biology, University of Texas negative stain electron microscopy show that each type of Medical School, Houston, TX 77030. complex forms particles of Ϸ90–160 Å (1 Å ϭ 0.1 nm) in © 2005 by The National Academy of Sciences of the USA

10482–10486 ͉ PNAS ͉ July 26, 2005 ͉ vol. 102 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503558102 Downloaded by guest on September 26, 2021 Fig. 2. Classiﬁcation of TatA complexes. (A) The untilted data were sorted into 10 classes according to size (28). The averages of all images in each class are shown. The number of images per class ranges from 86 (class 9) to 249 (class 3). (B) Averages of the untilted data in A after reference-free alignment. Four representative classes used in the interpretation of the 3D analysis are outlined.

Pairs of low-dose exposures were taken of each selected area, the Fig. 1. Characterization of TatA complexes. (A and B) Micrographs of TatA, first at a nominal tilt angle of 45° and the second at 0°. The stained with uranyl acetate. (A) Untilted specimen. (B) Forty-ﬁve-degree tilted ␮ view of the same area. The tilt axis runs horizontally. Corresponding particles average defocus was 1.5 m. Negatives were developed in Kodak are marked in each micrograph (white circles). A few large, collapsed vesicles, full-strength developer for 12 min, and their quality was assessed probably consisting of lipid and͞or detergent are also present. (C) SDS͞PAGE by optical diffraction. Micrographs were digitized on a SCAI analysis and Coomassie brilliant blue staining of the sample used for image microdensitometer (Zeiss) with a pixel size of 14 ␮m. analysis. The molecular masses (kDa) of marker proteins are given on the left. The band corresponding to TatA as well as the expected positions of TatB and Image Processing. Image analysis was performed by using TatC are indicated on the right. (D) Immunodetection of Tat proteins in the IMAGIC-5 (24) and SPIDER/WEB software (25). The random con- TatA sample used for image analysis. The sample was subjected to SDS͞PAGE ical tilt analysis (26) was based on SPIDER scripts written by N. followed by immunoblotting with the indicated subunit-speciﬁc antisera. Identical exposures were used in all three cases. (E) Blue native PAGE analysis Boisset (Universite´Pierre et Marie Curie, Paris). of the sample used for image analysis, stained by Coomassie brilliant blue. The Particles were picked interactively using the ‘‘tilted particles’’ molecular masses (kDa) of marker proteins are given on the left. Arrows on the option in WEB, which provided the tilt geometry for each right indicate bands corresponding to the variable-size TatA complexes negative. CTFTILT3 (27) was used to calculate the defocus in present in the sample. each tilted micrograph and to verify the tilt geometry found during particle picking. The particles were corrected for the effect of the contrast transfer function by phase-flipping in folded proteins while maintaining the ionic permeability barrier SPIDER according to their positions on the micrograph. of the membrane. The untilted particles were centered by multi-reference Experimental Methods alignment. Seven soft-edged discs with radii of 14–20 pixels were used as initial references for translational alignment. Protein Chemistry. E. coli TatA with a C-terminal hexahistidine Next, the centered images were sorted by size by using the tag was overproduced from plasmid pFAT75AH (13) in E. coli approach described by White et al. (28) and classified by using ⌬ ⌬ tatABCD tatE strain DADE (22) as described in ref. 13. TatA multivariate statistical analysis. Finally, the data set was complexes from the overproducing strain were purified in the separated into 10 classes of varying size containing between 86 detergent C12E9 by successive Ni(II)-affinity and size exclusion and 249 members (Fig. 2A). chromatography steps as described in ref. 14. Immunoblotting For each class, the untilted images were aligned translationally revealed that only trace amounts of TatB, not visible by Coo- and rotationally with a reference-free approach defining the massie brilliant blue staining, are present in the TatA sample in-plane rotation necessary to align the images. The correspond- whereas TatC is completely absent (Fig. 1 C and D). Blue native ing tilted images were centered by translational alignment only. PAGE analysis of the purified TatA complexes was performed The Euler angles are defined by the tilt geometry (out-of-plane as described in ref. 23. angles) and the in-plane orientation of each particle determined from the untilted views. With these Euler angles for each Electron Microscopy. For structural analysis, 5 ␮l of the isolated particle, 3D reconstructions were computed by back projection TatA complex (0.01 mg͞ml) in 20 mM Mops, pH 7.2͞200 mM by using the SIRT algorithm (29). ͞ NaCl 0.1% (vol/vol) C12E9 was applied to carbon-coated copper In some of the initial reconstructions, there was weak density grids (400 mesh), which had been freshly glow-discharged in a on the open face, suggesting that some of the particles had the pentylamine atmosphere. After rinsing the grid three times with opposite orientation on the support film. To refine the orien- 20 mM Mops, pH 7.2͞200 mM NaCl, the specimen was stained tations, the surface with the weaker density was masked to create twice with 2% (wt/vol) uranyl acetate. a more asymmetric model. Reprojections were calculated for 45° Micrographs were recorded using a Tecnai T10 electron tilts of this model and used to realign the original, tilted images

microscope (FEI, Eindhoven, The Netherlands) operated at an and refine the tilt direction by projection matching (29). The BIOCHEMISTRY accelerating voltage of 100 kV with a magnification of ϫ44,000. extra density did not reappear after this refinement, and the

Gohlke et al. PNAS ͉ July 26, 2005 ͉ vol. 102 ͉ no. 30 ͉ 10483 Downloaded by guest on September 26, 2021 Results Electron Microscopy of TatA. Electron microscopy of TatA complexes solubilized in the detergent nona-polyoxyethylene dode- cyl ether (C12E9) and negatively stained reveals mainly ring- shaped particles (Fig. 1A). Preliminary analysis indicated that the particles vary in size, that most adopt a preferred orientation, and that no symmetry could be identified. Therefore, we used a strategy of size separation followed by random conical tilt analysis (26) to obtain the different views required for 3D reconstruction of the complex. A micrograph of the area in Fig. 1A tilted at an angle of 45° is shown in Fig. 1B. The particles appear elliptical, with the long axis lying parallel to the tilt axis. Approximately 1,800 particles were selected from 11 pairs of tilted and untilted views. Multi- variate statistical analysis (MSA) of the untilted particles confirmed the presence of size variations. Using MSA, we sorted the particles into 10 size groups using the strategy described by White et al. (28). The class averages are shown in Fig. 2A. Their members (86–249 images per class) were aligned by using a reference-free approach (29) to determine the in-plane orientation of each particle (Fig. 2B). For each class, a 3D density map Fig. 3. Comparison of observed views with reprojections of the 3D maps. (A and B) Averages of the classes outlined in Fig. 2B (A) for comparison with was reconstructed by using the corresponding data from the reprojections of the corresponding 3D maps of TatA after reﬁnement (B). tilted specimen. The maps showed a cylindrical ring with addi- (C and D) Raw images of tilted views selected from different size groups (C) tional protein density forming a lid covering the central channel for comparison with reprojections of the 3D maps at the corresponding and less density on the opposite surface. Separate analysis of an orientations (D). independent sample (data not shown) confirmed the appearance of a cylindrical structure with a lid at one end only. The asymmetric exposure of protein at the two surfaces is also asymmetry was subsequently confirmed by an independent consistent with the results of protease accessibility studies of the analysis of a different data set, which showed clear asymmetry TatA complex in its native membrane environment (14). without the use of a masked reference. From the realigned After refinement by projection matching, the series of conical images, final 3D reconstructions were computed and filtered tilt reconstructions obtained from the set of different size classes to 25 Å. showed a corresponding range from small to large TatA com-

Fig. 4. The 3D architecture of assembled TatA complexes. Shown are four TatA complexes with increasing diameter. The 3D maps are ﬁltered between 150 Å and 25 Å and contoured at Ϸ4 ␴ (standard deviations above the mean density). (A) TatA complexes viewed from the closed end of the channel, proposed to be at the cytoplasmic side of the membrane (C-face; see text). Density forming the lid domain can be clearly seen. (B) TatA complexes viewed from the open end of the channel, proposed to be at the periplasmic side of the membrane (P-face). (C) Side views of TatA. The front half of each molecule has been cut away to reveal internal features. (D) Views of TatA parallel to the membrane plane. The proposed position of the lipid bilayer is indicated in blue. (Scale bar, 100 Å.) The ﬁgure was prepared with PYMOL (www.pymol.org).

10484 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503558102 Gohlke et al. Downloaded by guest on September 26, 2021 Table 1. Three-dimensional structure analysis of TatA complexes Class 1 Class 4 Class 8 Class 10

No. of particles 147 206 235 86 Outer diameter, Å 85–90 90–95 100–110 125–130 Channel diameter, Å 30–35 40–45 55–60 65–70 Total height, Å 55–60 65–70 75–80 80–85 Ring height, Å 45–50 45–50 50–55 50–55 Ring width, Å 25–30 25–30 25–30 25–30 Molecular mass, kDa 130–150 160–180 230–280 330–390 Subunit number, volume 12–14 15–17 21–25 30–35 Subunit number, circumference 19 21 26 31

Speciﬁcations for four classes are listed in ascending order of size. Dimensions and molecular masses are given for the range of contour levels from 3.5 to 4.5 ␴ and are rounded to the nearest 5 Å and 5 kDa, respectively. Outer diameter, complex in the orientation shown in Fig. 4B; Total height, orientation shown in Fig. 4D; Ring height, height of the protein density which is proposed to be membrane-spanning; Ring width, thickness of the ring wall as viewed in Fig. 4C. The range of molecular masses of each complex was estimated from the volume enclosed at contour levels of 3.5 and 4.5 ␴, assuming a protein density of 0.844 Da͞Å3. The number of TatA subunits was estimated either from the volume or based on transmembrane helix packing around the ring circumference. The ﬁrst estimate is obtained by dividing the complex molecular mass by that of the His6-tagged TatA subunit (10,730 Da), assuming that all residues are present in the observed volume. Alternatively, the number of TatA subunits is calculated from the average circumference (center of the ring wall), assuming a center-to-center distance of 10 Å for transmembrane helices.

plexes. For the following interpretation, we have chosen four in detergent solution as it does in the membrane, it is likely that classes representing the smallest and the largest complex as well the lid of TatA is located on the cytoplasmic side (C-face, Fig. as two intermediate sizes (Fig. 3A). The selected class averages 4A) while access to the pore on the periplasmic side is unre- are compared with the corresponding reprojections of the final stricted (P-face, Fig. 4B). 3D density maps in Fig. 3B. For the tilted views, the orientation of each is defined by the tilt geometry, so they cannot be grouped Discussion into class averages. In this case, individual tilted particles rep- The 3D architecture of the E. coli TatA complex shows that TatA resenting the size range (Fig. 3C) are compared with reprojec- forms a large transmembrane channel. In the largest of the TatA tions at the same orientations (Fig. 3D). complexes this channel is sufficiently wide (65–70 Å; Table 1) that it could allow passage of even the biggest E. coli Tat substrates (20). Three-Dimensional Architecture of the TatA Complex. The 3D density Therefore, the structural features strongly support the proposal that maps of all size classes of TatA show the same general archi- TatA is the protein-conducting channel of the Tat system. tecture, with a cylindrical ring of variable diameter enclosing a The lid region and variable diameter of the TatA complexes central channel (Fig. 4). One end of the channel is open, and the could explain how TatA is able to form a large transmembrane other shows extra density in a lid structure resulting in a height channel without compromising the membrane permeability barrier. of 55–85 Å for the TatA complexes (Table 1). The outer The lid structure could act as a gate, preventing solute access and diameters of the rings vary from 85 Å to 130 Å, and the pore size regulating substrate entry. In addition, it is notable that the range varies from 30 Å to 70 Å (Table 1). In all classes the wall of the Ϸ of variation in channel diameter between individual TatA com- ring has a roughly rectangular cross section 25–30 Å wide and plexes (30–70 Å; Table 1) matches the range of diameters found in 45–55 Å high (Fig. 4C and Table 1). Therefore, the variation in E. coli Tat substrates (20, 21). Therefore, the channel may change ring diameter (and mass) between classes is only due to differ- diameter to allow TatA to pack tightly around substrates of differing ences in the circumference of the rings and indicates that they size and prevent ion leakage during transport. As discussed in contain a variable number of subunits. This is supported by blue Results, the variation in diameter between TatA complexes arises native PAGE analysis of the preparation, indicating that suc- from variations in the number of their TatA subunits, which means cessive TatA oligomers vary in size by discrete steps of Ϸ40 kDa rather than in a continuous manner (Fig. 1E). Comparable that the changes in channel diameter required in a dynamic packing differences in TatA oligomer molecular weights have been mechanism must arise from addition or removal of protomers from reported by others for TatA solubilized in a different detergent the channel complex. (30). Each TatA protomer has a molecular mass of 10.7 kDa, The transmembrane portion of the TatA complex forms a ring Ϸ Ϸ suggesting that successive oligomers differ in composition by that is 45–55 Å high and 25–30 Å wide (Fig. 4C and Table more than a single TatA subunit. 1). Similar dimensions are found in the c10 ring of yeast ATP Because the TatA subunits must all be inserted into the synthase, in which the protein ring is 23–28 Å wide and 47–58 Å ␣ membrane in equivalent environments, the plane of the ring high and is formed by two transmembrane -helices linked by a must lie in the plane of the membrane. The 50-Å height of the loop (31). Depending on the contribution of the detergent ring is comparable with the thickness of a lipid bilayer. There- molecules to the density, this comparison suggests that the TatA fore, the structure is suitable to act as a channel for the ring is one or two transmembrane helices wide. The number of translocation of folded proteins across the membrane, with the TatA subunits forming the ring can be estimated from the lid domain controlling entry to this channel, as shown in Fig. 4D. circumference, assuming either a single ring of transmembrane We propose that the lid domain is on the cytoplasmic side on the helices or two concentric rings of helices, with one or two helices, basis of previous protease accessibility studies (14). TatA is not respectively, being contributed by each subunit. For a TatA accessible to digestion by proteinase K from the periplasmic side average diameter of 60–100 Å and a helix center-to-center

of the membrane, whereas it is readily degraded from the distance of 10 Å, this would give an estimate of 19–31 subunits BIOCHEMISTRY cytoplasmic side. Provided that TatA has the same conformation (Table 1).

Gohlke et al. PNAS ͉ July 26, 2005 ͉ vol. 102 ͉ no. 30 ͉ 10485 Downloaded by guest on September 26, 2021 TatA proteins are predicted to have a secondary structure in In conclusion, the observed size variation in TatA shows a which an N-terminal hydrophobic ␣-helix is followed by an remarkable flexibility in its structural organization and supports amphipathic ␣-helix and then a charged and largely unstructured the possibility that the Tat channel forms a close seal around the C-terminal tail (9, 32). This model for the secondary structure substrate protein during the transport process by adjusting the content is consistent with circular dichroism spectroscopy of number of TatA protomers in the channel complex (21). The TatA (14). Protein engineering experiments have demonstrated observed variation in the volume of the internal channel is that the N-terminal hydrophobic region up to residue 20 is sufficient to accommodate the variation in size of the Tat essential for both the membrane association and oligomerization substrate proteins. of TatA (14), compatible with the proposal that the N-terminal region forms a transmembrane helix. The TatA ring could be We thank Nicolas Boisset and Catherine Ve´nien-Bryan (University of formed by the N-terminal helices from each subunit or by a ring Oxford) for providing the SPIDER scripts for random conical tilt recon- of helical hairpins in which a second, amphipathic helix from struction and refinement as well as for their advice on the implemen- each subunit lines the channel, surrounded by an outer ring of tation; Luchun Wang for electron microscopy support; David Houlder- hydrophobic helices. The range of molecular masses of the TatA shaw, Steve Terrill, and Richard Westlake for computer support; Frank complexes estimated from the contoured volume of the density Sargent and Meriem Alami for their contributions to the characteriza- maps is 130–390 kDa (Table 1). Because the molecular mass of tion of TatA samples; George Orriss and Sonya Schermann (University the TatA protomer is 10,730 Da, the volume estimates suggest of Oxford) for providing additional TatA samples; and Jennifer Stewart Ϸ12–35 monomers per complex (Table 1), in good agreement for critical comments on the manuscript. This work was supported by with the results inferred from fitting helices into the transmem- Biotechnology and Biochemical Sciences Research Council Grant 83͞ brane ring, favoring the model in which amphipathic helices line B14749. Tracy Palmer is a Medical Research Council Senior Non- the channel. Clinical Research Fellow.

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