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• W2024490998 abstract "Article1 September 2003free access Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 Å resolution Johannes Furst Johannes Furst Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Department of Physiology, University of Innsbruck, Fritz-Pregl Straße 3, A-6020 Innsbruck, Austria Search for more papers by this author R.Bryan Sutton R.Bryan Sutton Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA, 94305-5432 USA Present address: Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX, 77555-0641 USA Search for more papers by this author James Chen James Chen Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Search for more papers by this author Axel T. Brunger Corresponding Author Axel T. Brunger Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA, 94305-5432 USA Search for more papers by this author Nikolaus Grigorieff Corresponding Author Nikolaus Grigorieff Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Search for more papers by this author Johannes Furst Johannes Furst Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Department of Physiology, University of Innsbruck, Fritz-Pregl Straße 3, A-6020 Innsbruck, Austria Search for more papers by this author R.Bryan Sutton R.Bryan Sutton Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA, 94305-5432 USA Present address: Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX, 77555-0641 USA Search for more papers by this author James Chen James Chen Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Search for more papers by this author Axel T. Brunger Corresponding Author Axel T. Brunger Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA, 94305-5432 USA Search for more papers by this author Nikolaus Grigorieff Corresponding Author Nikolaus Grigorieff Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA Search for more papers by this author Author Information Johannes Furst1,2, R.Bryan Sutton3,4, James Chen1, Axel T. Brunger 3 and Nikolaus Grigorieff 1 1Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, MA, 02454 USA 2Department of Physiology, University of Innsbruck, Fritz-Pregl Straße 3, A-6020 Innsbruck, Austria 3Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H.Clark Center, E300-C, 318 Campus Drive, Stanford, CA, 94305-5432 USA 4Present address: Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX, 77555-0641 USA ‡J.Furst and R.B.Sutton contributed equally to this work *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2003)22:4365-4374https://doi.org/10.1093/emboj/cdg420 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP–ADP mixture at 11 Å resolution by electron cryomicroscopy and single-particle averaging of NSF·α-SNAP·SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to α-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF. Introduction In eukaryotic cells, trafficking of vesicles is essential for diverse cellular functions such as exocytosis, endocytosis, transcytosis and subcellular compartmentalization (Rothman, 1994). NSF belongs to the AAA (ATPases Associated with a variety of cellular Activities) family of ATPases and is an essential component of the protein machinery that regulates vesicle fusion with target membranes. NSF acts together with α-SNAP (Soluble NSF Attachment Protein) to disassemble SNARE (SNAP REceptor) complexes. SNAREs, in a concerted action with additional proteins, facilitate docking and fusion of vesicles, and they are recycled and reactivated through disassembly by NSF (Brunger, 2001b; Whiteheart et al., 2001). NSF also interacts with other membrane proteins, such as glutamate [GluR2 (Nishimune et al., 1998), AMPA (Braithwaite et al., 2002)], GABA (Kittler et al., 2001) and β2 adrenergic receptors (Cong et al., 2001), indicating a possible additional role in trafficking of membrane proteins. In its functional state, NSF is a homohexamer (Fleming et al., 1998). Each of the NSF protomers contains three domains. The N-terminal domain of NSF (NSF-N) is essential for the binding of α-SNAP and is followed by ATPase domains D1 and D2. Binding and hydrolysis of ATP by the NSF-D1 domain induces conformational changes in NSF leading to disassembly of the SNARE complex. NSF-D2 has a lower ATPase activity than NSF-D1. ATP probably acts as a structural component for NSF-D2, since its binding is required for the hexamerization of NSF (Yu et al., 1998; Whiteheart et al., 2001). Electron microscopy (EM) of NSF and NSF·α-SNAP·SNARE (20S) complexes has provided a basic understanding of the overall structure of NSF and 20S complexes at low resolution, as well as demonstrating possible conformational changes of the NSF hexamer during ATP hydrolysis (Hanson et al., 1997; Hohl et al., 1998). Of the three NSF domains, crystal structures of NSF-N (May et al., 1999; Yu et al., 1999) and hexameric NSF-D2 (Lenzen et al., 1998; Yu et al., 1998) have been obtained, but there are no three-dimensional (3D) structures available for the D1 domain or the complete NSF molecule, and consequently the quarternary arrangement of the NSF domains is uncertain. Here we present the 3D structure of the hydrolysis mutant E329Q of NSF complexed with α-SNAP, SNAREs and a mixture of ATP and ADP at 11 Å resolution, determined by electron cryomicroscopy (cryo-EM) and single-particle averaging. The hydrolysis mutant was used to slow ATP turnover and help stabilize a single conformation of NSF. The NSF domains have been assigned by imaging of truncated NSF constructs in negative stain and by docking of the NSF-D2 crystal structure into the 3D density map. Results As a prelude to cryo-EM, the E329Q mutant of NSF (NSFm) (Whiteheart et al., 1994) and the complex of NSFm, α-SNAP and SNAREs (20Sm) were studied by negative stain EM. The initial studies using negatively stained protein served to determine the location of NSF domains within the 20Sm complex, which assisted in the interpretation of the density map obtained by cryo-EM at higher resolution. NSFm in negative stain NSFm was purified, applied to carbon-coated copper grids and negatively stained with methylamine tungstate or uranyl acetate as described in Materials and methods. Similar to recombinantly expressed wild-type NSF (Hanson et al., 1997; Hohl et al., 1998), single particles of NSFm adopt a preferred orientation with a donut-like appearance and 6-fold symmetry on carbon support films. This orientation will henceforth be referred to as the 6-fold view. Other views that are only infrequently detected in uranyl acetate, but more regularly observed in methylamine tungstate, show the typical barrel-shaped double-layered structure observed for NSF (Hohl et al., 1998). These views will be referred to as side views. Small arms, corresponding to the globular density detected in 6-fold views of NSFm, extend at various angles upwards from only one of the two layers (Figure 1A and B). Deletion of the N-terminal domain of NSF removes these densities (Figure 1C), establishing that the double-layered barrel contains the two ATPase domains NSF-D1 and NSF-D2, while the N-terminal NSF-N domain is flexible and extends sideways and upwards from the barrel. Figure 1.NSFm and 20Sm complexes in negative stain. (A) NSFm in 1 mM ADP stained with 2% methylamine tungstate. Left: side views show the typical double-layered barrel described for wild-type NSF (Hohl et al., 1998). Right: 6-fold views are donut-shaped particles from which density extends sideways (arrows). Arm-like densities (arrows) corresponding to the NSF-N domain extend at various angles from one end of the double-layered NSFm barrel. (B) NSFm in 1 mM ATPγS stained with 2% methylamine tungstate. Single particles of NSFm in ATPγS are indistinguishable from single particles of NSFm in ADP. Arrows point to the flexible NSF-N domains. Left: side views. Right: 6-fold views (C) NSF-N deletion mutant (NSFΔN) in 1 mM ADP stained with 2% methylamine tungstate. Although the overall structure of NSFΔN is similar to that of NSFm in either ADP or ATPγS, no densities extending sideways or upwards from NSF can be observed, indicating that the protrusions seen in NSFm correspond to the NSF-N domain, while the double-layered barrel contains the NSF domains D1 and D2. Left: side-views. Right: 6-fold views (D) 20Sm complexes in 0.1 mM ATP/0.01 mM ADP stained with 1% uranyl acetate show all features described for wild-type 20S complexes (Hohl et al., 1998), i.e. the barrel-shaped NSFm particle that carries a cone-shaped complex at one end. Scale bar, 100 Å. Download figure Download PowerPoint 20Sm complexes in negative stain Similar to NSFm in negative stain, 20Sm complexes consisting of NSFm, a truncated SNARE complex fused to MBP and α-SNAP adopt a preferred orientation on the carbon support film. Six-fold views (Figure 1D) were prominent and side views were only infrequently observed, regardless of the negative stain used (uranyl acetate or methylamine tungstate). These 6-fold views appear indistinguishable from 6-fold views of NSFm (compare Figure 1A, B and D). However, class averages of side views (height ∼190 Å, width ∼130 Å) could be obtained and show the features previously described for complexes of wild-type 20S complexes (Hohl et al., 1998), i.e. a double layered NSF barrel (height ∼105 Å, width ∼130 Å) and a cone-shaped complex ∼80 Å long, corresponding to α-SNAP and SNAREs (Figure 1D). The cone-shaped extension in the images shown by Hohl et al. (1998) is more pronounced than in our images since they used full-length SNARE proteins, including the transmembrane domains which were bound to detergent micelles. 20Sm by electron cryomicroscopy 20Sm was frozen in vitreous ice and examined at liquid nitrogen temperature under low dose conditions (∼10 e−/A2). Unlike NSFm (data not shown), 20Sm complexes produced high contrast in ice (Figure 2A), enabling the selection of a total of 31 592 images of single particles from digitized micrographs. An initial 3D density map of 20Sm with imposed 6-fold symmetry was calculated from a subset of 10 002 particle images using the IMAGIC image processing package (van Heel et al., 1996), as described in Materials and methods. Class averages obtained in the final round of classification, using IMAGIC, correspond well to matching projections of the 3D structure (Figure 2B). This initial 3D density map of 20Sm was subsequently further refined to 20 Å resolution and corrected for the contrast transfer function (CTF) of the electron microscope using FREALIGN (Grigorieff, 1998) (Figure 2C). Figure 2.Initial 3D structure of 20Sm complexes in ice. (A) 20Sm particles produce high contrast in ice. Scale bar, 200 Å. (B) Class averages (first row) of selected views of 20Sm complexes demonstrating good correspondence with matching projections (second row) from the 3D density map calculated by angular reconstitution as implemented in IMAGIC. The third row shows 3D surface views with orientations corresponding to the projections in the second row. Scale bar, 100 Å. (C) Preliminary 3D density map of 20Sm. Left: NSFm forms a double-layered barrel. Protrusions, similar to those observed in NSFm in negative stain, extend sideways from one end of the barrel. Near the protrusions, a cap-like density that coincides with the location of α-SNAP and SNARE in 20Sm complexes in negative stain is clearly visible around the 6-fold symmetry axis. The protrusions fuse with disconnected density next to the cap, when the molecular weight of the structure is increased, indicating flexibility in this region. Right: the reasonably good fit of the NSF-D2 crystal structure (PDB entry code, 1NSF.pdb) into both layers of the NSFm barrel demonstrates that the major density in 20Sm contains NSF-D1 and NSF-D2. For these fits, we assumed an antiparallel arrangement of the D1 and D2 domains (see text). Download figure Download PowerPoint As shown in Figure 3C for the entire data set of 31 592 particles (see below), Euler angles of 20Sm particles sample the entire 3D space, with a slight preference for 6-fold and side views, allowing a reliable 3D reconstruction of 20Sm. To interpret the initial 20Sm density map, the crystal structure of NSF-D2 (Yu et al., 1998) (PDB entry code: 1NSF.pdb) was manually docked into the density map of 20Sm. The resulting model produced good agreement for both layers of the barrel-like density of 20Sm, demonstrating that the reconstruction procedure described above produced a reliable density map in the region of the two NSF ATPase rings (Figure 2C). Figure 3.20Sm in ice at 11 Å resolution: Fourier shell correlation (FSC) and distribution of Euler angles. (A) Right: side view and the two end views of the structure (scale bar, 100 Å). Left: analysis of the variance in the 20Sm density map. The variance increases towards the end of the structure that is close to the NSF-N domains of 20Sm, indicating considerable flexibility in this region. (B) FSC indicates a resolution of 13.5 Å at a correlation value of 0.5 for the 20Sm structure with tilt-corrected defocus values. (C) 20Sm particles in ice sample the entire 3D space with a slight preference for end and side views. Download figure Download PowerPoint 20Sm at 11 Å resolution Inclusion of all 31 592 single particles and further refinement using FREALIGN produced a density map at 11 Å resolution (see Materials and methods). Similar to the initial 20Sm density map described above, the 11 Å density map of 20Sm is characterized by a double-layered barrel that measures ∼120 Å along the symmetry axis and has a diameter of ∼130 Å. At one end of 20Sm, six protrusions emerge (top of the side view in Figure 3A) and a protruding feature located on the 6-fold axis of the density map is visible. The location of the six protrusions coincides with the location of the NSF-N domains, as evidenced by the low-resolution images of the NSF N-terminal deletion mutant determined in negative stain (Figure 1C). The density on the 6-fold axis overlaps with density attributed to α-SNAP and SNAREs in Figure 1D. The NSF-N domains, α-SNAP and SNAREs are not clearly resolved in the density map. Since the SNARE complex does not possess 6-fold symmetry, one expects to lose structural detail in this part of the complex upon imposition of 6-fold symmetry. The unresolved density may also be due to a variable number of SNAREs present in the 20S complexes. However, the loss of structural detail in the region of the NSF-N domains is most likely due to flexibility in this part of the molecule. The lower quality of the density map in the region of NSF-N is also indicated by an increased density variance (Figure 3A). Docking of the NSF-D2 crystal structure The crystal structure of NSF-D2 complexed with ATP (Yu et al., 1998) (PDB entry code: 1NSF.pdb) was docked into the 11 Å EM density map both manually and using SITUS 2.0 (Wriggers et al., 1999). Consistent with visual inspection and manual docking, the volumetric docking algorithm of SITUS 2.0 placed the crystal structure of NSF-D2 into the layer of the EM density map that is opposite to the one with the six protrusions (Figure 4). When fitted into 20Smbot, the crystal structure produced the highest correlation coefficient (0.86) with the C-terminus and the helical subdomain facing towards the interface between the two ATPase domain rings (Figure 4A, panels 1–4). The unusually high quality of the fit is illustrated, for example, by the good agreement of two density features at the perimeter of the D2 ring with two α-helices of the helical subdomain of NSF-D2 (Figure 4A, panel 3, arrows). Placement of the D2 ring in the opposite orientation, i.e. with the helical subdomains pointing away from the other NSF domains, produced a lower correlation coefficient (0.79) and there were clear discrepancies between the crystal structure and the 20Sm density map (Figure 4B, arrow). We interpret residual discrepancies between the fitted crystal structure of NSF-D2 in the orientation shown in Figure 4A (panels 1–4) and the EM map as an indication of small conformational differences between the crystallized NSF-D2 structure and the EM density map. Figure 4.Docking of the NSF-D2 crystal structure into 20Smbot. (A) Docking of the NSF-D2 crystal structure (PDB entry code, 1NSF.pdb) by SITUS 2.0 (Wriggers et al., 1999) reveals good agreement with the layer distal to the protrusions of 20Sm (20Smbot, bottom of panel 4). Panels 1–3: slices through 20Smbot perpendicular to the symmetry axis starting from the bottom of the structure. Arrows point to two α-helices of the C-terminal helical NSF-D2 subdomain that fit remarkably well into two separated densities in 20Smbot. The separation between these two α-helices suggests a resolution of the 20Sm structure of 11 Å. Panel 4: slice parallel to the symmetry axis through 20Sm. The best fit of NSF-D2 places it into 20Smbot, such that the N-terminus of NSF-D2 faces away from the interface with 20Smtop and the C-terminal helical NSF-D2 subdomain faces toward it. (B) Slice corresponding to panel 4 through 20Sm with NSF-D2 rotated 180° around an axis perpendicular to the symmetry axis and docked into 20Smbot. Considerable mismatch between NSF-D2 and the density map of 20Sm can be observed (arrow). Download figure Download PowerPoint Comparison of 20Smbot and 20Smtop by correlation analysis No detailed structural information on NSF-D1 is available as it has not been crystallized so far. AAA ATPase domains are generally believed to be structurally homologous, especially for the α/β subdomain (Ogura and Wilkinson, 2001). However, larger differences have been observed for the helical subdomain (DeLaBarre and Brunger, 2003). Since NSF-D1 and NSF-D2 are structurally related, one should expect some structural similarity between the two density regions in the EM density map designated as 20Smbot and 20Smtop (see above and Figure 6A). To obtain further insight into the similarity between these two density regions, and to determine the orientation of the NSF-D1 domain with respect to NSF-D2, the 20Smbot density was rotationally and translationally fitted into the 20Smtop density by maximizing a correlation coefficient. This approach represents a model-free analysis of the arrangement of the two ATPase domains within NSF. Two different orientations were tested: the parallel orientation, in which 20Smbot is shifted into the 20Smtop density, and the antiparallel orientation, in which 20Smbot is rotated by 180° around an axis perpendicular to the 6-fold symmetry axis and shifted into the 20Smtop density, as depicted in Figure 6. For each orientation of 20Smbot, a rotational search around the 6-fold symmetry axis was carried out, and the rotation angle with the best correlation coefficient was chosen for further analysis. In the parallel orientation of 20Smbot and 20Smtop, the correlation coefficient is 0.47, and the outer surface of 20Smbot agrees well with the outer surface of 20Smtop over the entire superimposed area, including the transition zone to the protrusions on the side of 20Smtop (Figures 5, panels A1–A5 and 6B, left panel). However, internal surfaces show considerable density mismatches, especially at the interface between 20Smbot and 20Smtop (Figure 5, panels A1 and A2, arrows) and in the area close to the protrusions on 20Smtop (Figure 5, panels A4 and A5, arrows). To illustrate the differences between the two densities further, the aligned 20Smbot density was subtracted from 20Smtop to produce a difference map. A two-dimensional (2D) projection of this difference map is shown as an inset in Figure 6B, left panel. Apart from large difference peaks near the putative locations of the N domains, there are also strong peaks at the interface between 20Smbot and 20Smtop. Figure 5.Comparison of 20Smbot and 20Smtop. (A) Comparison of 20Smbot with 20Smtop arranged in a parallel orientation (see also Figure 6). 20Smbot (red) was rotationally and translationally fitted into 20Smtop (yellow) as described in the Results and shown in Figure 6. Panels A1–A5: slices through 20Smtop perpendicular to the symmetry axis, starting at the interface between 20Smbot and 20Smtop. Arrows point to densities that do not overlap in 20Smtop and 20Smbot. Panels A6–A10: the transformation of the best parallel fit of 20Smbot into 20Smtop was used to dock NSF-D2 into 20Smtop. The slices through 20Smtop correspond to panels A1–A5. It is evident from panels A6 and A7 that the NSF-D2 crystal structure does not agree well with 20Smtop near the interface with 20Smbot. In the transition zone toward the protrusions on 20Smtop (panels A8 and A9), NSF-D2 shows better agreement with 20Smtop. For this fit, the C-terminal α-helical NSF-D2 subdomain extends upward into the protrusions at one end of 20Smtop (panel A10). The arrows point to density that is not occupied by 20Smbot and the NSF-D2 crystal structure; the + signs in panels 6 and 7 show the NSF-D2 α-helices that do not fit into the 20Smtop density. (B) Comparison of 20Smbot with 20Smtop arranged in the antiparallel orientation. 20Smbot (blue) was rotationally and translationally fitted into 20Smtop (yellow) as described in the Results. Panels B1–B5: slices through 20Smtop perpendicular to the symmetry axis, starting at the interface between 20Smbot and 20Smtop. Arrows point to non-overlapping density. Panels B6–B10: the transformation of the best antiparallel fit of 20Smbot into 20Smtop was used to dock NSF-D2 into 20Smtop. The slices through 20Smtop correspond to panels B1–B5. NSF-D2 agrees well with the 20Sm density map throughout the entire volume. All major densities overlap. The arrows point to non-overlapping densities. Download figure Download PowerPoint Figure 6.Parallel versus antiparallel arrangement of NSF-D1 and NSF-D2. (A) 20Sm structure with 20Smtop in yellow and 20Smbot in blue. The arrow points in the direction of the rotational and translational fit carried out to compare 20Smtop with 20Smbot, and to determine the orientation of NSF-D1 with respect to NSF-D2. (B) Slice through 20Sm parallel to the symmetry axis. In the parallel arrangement of 20Smbot and 20Smtop, considerable density mismatch can be observed. The arrows point to density that does not overlap. Protrusions corresponding to the NSF-N domains are partially occupied. The inset shows a 2D projection of the difference map between 20Smbot and 20Smtop. Apart from the peaks in the region of the N domains, there are also strong peaks at the D1–D2 interface. In the antiparallel arrangement 20Smbot (blue) fits well into 20Smtop without major discrepancies between these two densities. For this arrangement, protrusions corresponding to the NSF-N domain remain unoccupied and are marked by an arrow. The difference map in the inset shows strong peaks in the region of the N domains, but not at the D1–D2 interface. (C) Fit of the NSF-D2 crystal structure into both 20Smbot and 20Smtop. The discrepancy seen between the parallel fit of the 20Smbot density into the 20Smtop density can also be seen between the NSF-D2 crystal structure and 20Smtop density. The arrows indicate density occupied in the antiparallel fit that is not occupied in the parallel fit. (D) Slice through 20Sm (yellow) at the interface between 20Smbot and 20Smtop, perpendicular to the symmetry axis. The position of the slice is marked by square brackets ] [ in (C). The NSF-D2 crystal structure fitted into the 20Smbot density is shown in light blue, and the NSF-D2 crystal structure fitted into the 20Smtop density is shown in dark blue or red for the antiparallel or parallel arrangement, respectively. The interface between 20Smbot and 20Smtop is well structured. For the antiparallel arrangement, almost the entire density is occupied and no significant discrepancies can be observed between the NSF-D2 crystal structure in 20Smbot and the NSF-D2 crystal structure in 20Smtop. The arrow points to a well-refined tube-like density that is likely to contain the N-terminus of NSF-D2 and/or the linker between NSF-D1 and NSF-D2. In the parallel arrangement, the NSF-D2 crystal structure does not fit well into 20Smtop at the interface between 20Smbot and 20Smtop. An α-helix of the NSF-D2 crystal structure that does not have a corresponding density in 20Sm is marked by a + sign. The arrows point to unoccupied density. (E) NSF-D2 crystal structures arranged in the parallel and antiparallel orientations. Download figure Download PowerPoint In the antiparallel orientation, the best correlation coefficient between 20Smbot and 20Smtop is 0.59, significantly better than that found for the parallel orientation. The internal surfaces of 20Smtop and 20Smbot are in good agreement throughout the entire overlapping area, as can be seen from the side view in Figure 6B, right panel and the perpendicular slices in Figure 5, panels B1–B5. Although all major densities of 20Smtop overlap with corresponding densities in 20Smbot, discrepancies in the overall appearance and location of these densities (Figure 5, panels B1–B5) are indicative of somewhat different conformations of the helical and α/β subdomains of NSF-D1 compared with those of NSF-D2, and unaccounted density corresponding to NSF-N. A density mismatch near the N domain would be consistent with a difference in the conformation of the N-terminus of NSF-D1 and that of the N-terminus of NSF-D2. The N-terminus of NSF-D1 is part of the linker to NSF-N and is likely to undergo considerable conformational changes along with NSF-N during ATP hydrolysis. As before, a difference map between 20Smbot and 20Smtop was calculated, and a projection is shown as an inset in Figure 6B, right panel. Unlike the parallel arrangement, the antiparallel arrangement of the two ATPase domains does not produce strong difference peaks at the domain interface. The main differences occur on the opposite side of the D1 domain, near the putative locations of the N domains. Fitting of the NSF-D2 crystal structure into 20Smtop The observed differences between the two ‘barrel’ densities, 20Smtop and 20Smbot, prevented straightforward fitting of NSF-D2 into 20Smtop by automated docking. In order to obtain a fit of NSF-D2 into 20Smtop, we used the transformations found for the best parallel and antiparallel alignments of 20Smbot to 20Smtop, as described above, to fit the NSF-D2 crystal structure, previously aligned to 20Smbot, into 20Smtop. The modeling of the 20Smtop density by the NSF-D2 crystal structure is clearly approximate since there may be differences between the NSF-D1 and NSF-D2 domains, especially in the helical domains. For the parallel fit, considerable density at the interface between 20Smbot and 20Smtop remains unoccupied, and an α-helix of the α/β subdomain extends into empty space inside the pore of the barrel (" @default.
• W2024490998 created "2016-06-24" @default.
• W2024490998 creator A5088336409 @default.
• W2024490998 date "2003-09-01" @default.
• W2024490998 modified "2023-10-16" @default.
• W2024490998 title "Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution" @default.
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