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Structure of the mitochondrial ATP synthase by electron cryomicroscopy

2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês

10.1093/emboj/cdg608

1460-2075

John L. Rubinstein,

RNA modifications and cancer

Article1 December 2003free access Structure of the mitochondrial ATP synthase by electron cryomicroscopy John L. Rubinstein Corresponding Author John L. Rubinstein MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6 Canada Search for more papers by this author John E. Walker John E. Walker MRC Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY UK Search for more papers by this author Richard Henderson Richard Henderson MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author John L. Rubinstein Corresponding Author John L. Rubinstein MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6 Canada Search for more papers by this author John E. Walker John E. Walker MRC Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY UK Search for more papers by this author Richard Henderson Richard Henderson MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information John L. Rubinstein 1,2, John E. Walker3 and Richard Henderson1 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK 2Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6 Canada 3MRC Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6182-6192https://doi.org/10.1093/emboj/cdg608 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 Å resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic α–β interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis. Introduction The mitochondrial adenosine triphosphate synthase (ATP synthase) is a membrane-bound multisubunit enzyme that couples the proton motive force across the inner mitochondrial membrane to the synthesis of ATP in the matrix (Boyer, 1997; Walker, 1998). The bovine complex contains 16 different proteins (including the regulatory subunit, IF1) and has a mass of ∼600 kDa (Walker et al., 1991). The extramembranous F1 catalytic subcomplex is attached to the membrane intrinsic FO subcomplex by a central stalk and a peripheral stalk (Walker, 1998). As protons pass through the membrane domain, a rotational force is generated in FO between the ring of c-subunits and the a-subunit. The rotational force causes the entire central rotor, the γδϵ-c10 subcomplex, to turn (Sambongi et al., 1999; Stock et al., 1999; Pänke et al., 2000) and drives the synthesis of ATP in the α3β3 subcomplex of F1. The enzyme can also catalyse the reverse reaction, with ATP hydrolysis in F1 driving proton pumping in FO. The peripheral stalk acts as a stator that prevents the α3β3 subcomplex from following the rotation of the central stalk. Consistent with this role, the peripheral stalk can be cross-linked to an α-subunit without preventing rotation of the rotor (Ogilvie et al., 1997). The peripheral stalk is composed of single copies of the oligomycin sensitivity conferral protein (OSCP) and subunits b, d and F6 (Collinson et al., 1994a, 1996). Residues 1–10 of OSCP (Joshi et al., 1996) interact with residues 1–15 of one or more α-subunits and with residues 1–5 of one or more β-subunits that are located at the top of F1 distal from FO (Hundal et al., 1983; Dupuis and Vignais, 1985; Walker et al., 1985; Abrahams et al., 1994). The OSCP extends almost 100 Å along the surface of F1 towards FO (Rubinstein and Walker, 2002). A C-terminal segment of OSCP interacts with the C-terminal domain of subunit b (Joshi et al., 1996), which continues towards the membrane. Subunits d and F6 both interact with subunit b (Collinson et al., 1994a). Residues 20–80 of subunit b probably form two transmembrane helices joined by a hydrophilic loop that protrudes into the intermembrane space (Walker et al., 1987). Since the OSCP-bdF6 subcomplex was proposed to form a separate domain and play the role of a stator (Walker, 1998), the peripheral stalk has been observed by electron microscopy (EM) in the isolated enzyme from various species (Böttcher et al., 1998; Wilkens and Capaldi, 1998a; Karrasch and Walker, 1999). The structures of several subcomplexes of ATP synthase have been determined by X-ray crystallography, most notably the F1-ATPase (α3β3γδϵ) from the bovine enzyme (Gibbons et al., 2000), the F1-c10 subcomplex (α3β3γδϵ-c10) from the yeast Saccharomyces cerevisiae (Stock et al., 1999) and the F1-ATPase (α3β3γ) from spinach chloroplasts (Groth and Pohl, 2001). The structure of the bovine inhibitor protein, IF1, has also been determined in isolation (Cabezón et al., 2001) and in complex with the F1-ATPase (Cabezón et al., 2003). In addition, the structures of several individual subunits and fragments of subunits have been solved by solution NMR spectroscopy. They include residues 1–105 of the Escherichia coli δ-subunit (Wilkens et al., 1997), residues 1–120 of the homologous bovine OSCP (R.J.Carbajo, F.A.Kellas, M.J.Runswick, J.E.Walker and D.Neuhaus, manuscript in preparation), the bovine F6-subunit (R.J.Carbajo, J.A.Silvester, M.J.Runswick, D.Neuhaus and J.E.Walker, manuscript in preparation), and residues 1–34 (Dmitriev et al., 1999) and 62–122 (Del Rizzo et al., 2002) of the E.coli b-subunit. However, there is still little information about how the subunits of the peripheral stalk interact with each other and with the F1 and FO regions of the enzyme. In this paper, we present images of the ATP synthase obtained by electron cryomicroscopy (cryoEM) of single particles. Class-averages derived from multivariate statistical analysis of these images reveal new structural information about both the FO membrane region and the peripheral stalk, and we provide evidence that these class-averages are authentic projected views of the complex. Using a new procedure, the views were placed in a sequence to form a ‘movie’ showing the complex as it is rotated about its long axis. The projected views of the structure were used to construct and refine a 3-D model for the ATP synthase that differs significantly from previously proposed models. An atomic model of the F1-c10 subcomplex has been docked unambiguously into a major segment of the EM map. This docking allows clear identification of the remaining density corresponding to the peripheral stalk and attached second domain in the FO membrane region. The structure suggests how the stator functions and provides significant restraints on the subunit organization in the FO region of the complex. Results Electron microscopy of the ATP synthase Using cryoEM, the bovine ATP synthase was imaged unstained in vitreous ice in holes in a perforated carbon support attached to a copper EM grid (Figure 1A). The images of the ATP synthase are, in general outline, similar to those obtained for the negatively stained molecule from a variety of sources (Böttcher et al., 1998; Wilkens and Capaldi, 1998a; Karrasch and Walker, 1999; Rubinstein and Walker, 2002). The images show two globular regions of different size connected by a central stalk. In most images, the globular F1 could be distinguished readily from the smaller, flatter, FO but it was not possible to identify the peripheral stalk unambiguously (Figure 1B). Figure 1.EM of ATP synthase particles. Frozen ATP synthase particles were imaged with a 200 kV electron microscope. (A) A sample region from a micrograph. (B) Some typical particle images. The scale bar in (A) represents 200 Å and the scale bar in (B) represents 100 Å. Download figure Download PowerPoint A dataset of 5984 particle images was aligned, subjected to multivariate statistical analysis (van Heel et al., 1996) and separated into 20 classes. Figure 2A shows the average of all 5984 particle images after alignment and Figure 2B–U shows the class-averages. In contrast to the strong orienting forces exerted on particles when they are attached to a continuous carbon support, particles in the thin film of buffer in a hole in a perforated carbon support are relatively free to adopt any orientation. However, there is a predominance of class-averages where the long axis of the complex lies almost parallel to the support. This observation indicates that either the thickness of the buffer film is less than the particle height of ∼200 Å or that there is some interaction between the particles and the air–water interface. Some portion of the thickness of the buffer film must be taken up by a monolayer of detergent at each air–water interface of the film, further reducing the height available to the protein particles. Figure 2.Class-average images of the ATP synthase. (A) Average of all of the aligned images in the dataset. The scale bar represents 100 Å. (B–E) Class-averages that probably arise from incoherently averaged particle images (and consequently, like the overall average, have a line of symmetry). (F–M) Class-averages where a corresponding mirror image is found (e.g. F with J, G with K, etc.) and constitute views about the long axis of the complex. (N and O) A mirror pair, but each probably consisting of averages of two non-equivalent views of the assembly with the long axis tilted from the plane of the grid. (P–U) Class averages that have no mirror pair in the dataset. Download figure Download PowerPoint The presence of mirror-image pairs in the class-averages can help to distinguish bona fide views of the particle from possibly artefactual class-averages that result from averaging non-equivalent or misaligned images. The two air–water interfaces of the buffer film should be indistinguishable from the point of view of the ATP synthase molecules. Therefore, if there were any interactions between the particle and the interfaces, an equal number of particles would be expected to interact with each interface. In the electron micrographs, particles interacting equivalently with opposite air–water interfaces would appear in projection as mirror images of each other. The consequence of this logic is that, whether or not there are interactions with air–water interfaces producing preferred orientations, mirror-image pairs in the class-averages should occur for authentic views. Indeed, even though images were never mirrored in the data analysis process, there exist 10 class-averages (F–O) that appear to have a mirror-image class-average in the data set (e.g. F with J, G with K, etc.). Class-averages F–M have the same overall length and are views of the particle where its long axis is parallel to the plane of the EM grid. In several of these views (H, I, L and M), the peripheral stalk of the complex is apparent. The nature of views F, G, J and K, where the peripheral stalk does not form a visible feature, cannot be interpreted sensibly without considering the 3-D structure of the enzyme and will be discussed later. The asymmetry of the FO region of the complex is also evident, and in class-averages G, H, K and L, FO appears to consist of two domains. The class-averages in the mirror pair of N and O are shortened relative to F–M, as if the long axis of the complex is not perfectly parallel to the plane of the grid. However, there are two non-equivalent ways in which the long axis of the complex can be tilted from the plane of the grid that would result in the same extent of shortening. Therefore, it is likely that both images N and O consist of a mixture of non-equivalent out-of-plane views. In contrast, several class-averages have no mirror images (Figure 2P–U). These class-averages should be interpreted with caution as they may be artefactual. Other class-averages (Figure 2B–E) appear to have a mirror line of symmetry. These symmetric class-averages are similar to the average of all of the images in the data set (Figure 2A) and therefore may also not correspond to views of the complex. Mirror lines are likely to arise by incorrectly classifying together and incoherently averaging particle images that actually consist of +90° and −90° rotations about an axis parallel to the grid. Consequently, the average of the class is symmetric, just as the average of all of the images in the data set is symmetric. The models proposed by several others (Wilkens, 2000; Mellwig and Böttcher, 2001; Mellwig and Böttcher, 2003) have incorporated mirror-symmetric class-averages as views, and thus may have forced the 3-D model to have a mirror plane of symmetry. It is important to note that the mirror-image logic does not apply to the case of particles attached to a carbon support. In that instance, the buffer layer has a unique air–water interface and a unique carbon–water interface, so that asymmetric views without a corresponding mirror-symmetric view may occur. Considering the significant asymmetry apparent in our side-views of ATP synthase, the question arises as to why such asymmetric views have not been apparent in the previously published images of the negatively stained complex. To answer this question, we prepared samples where ATP synthase particles were allowed to adsorb to a thin continuous carbon support, briefly rinsed and then vitrified (results not shown). This protocol mimics experiments done previously with negative stain, except that the frozen hydrated protein can be compared directly to our results for frozen hydrated ATP synthase in holes in a perforated carbon support-covered grid. After image analysis, it became apparent that adsorption to the carbon support introduced a further degree of preferred orientation to the particles. Mirror pairs in the class-averages did not exist for this set of images. No views were found where the domains of FO could be clearly observed or where the portion of the peripheral stalk that interacts with F1 did not overlap with F1. This strongly preferred orientation, with the peripheral stalk always on one side of the projection map, has been reported previously (Karrasch and Walker, 1999). Our observations offer a possible explanation for the limited orientations observed on continuous carbon supports. We cannot explain why Mellwig and Böttcher (2003), using perforated carbon, did not observe side-views showing two domains of FO in the chloroplast ATP synthase. Views of the complex arranged into a rotational sequence Class-averages in Figure 2F–M appear to constitute a single-axis rotation series with the rotation axis parallel to the y-axis of each image. This situation is similar to a tomographic series, but with unknown relative tilt angles. The common lines or angular reconstitution algorithms (Crowther et al., 1970; van Heel, 1987) are frequently used to determine the relative orientations of views of a molecule. However, for these methods to define the relative orientation of two views about their common line, a third view is required that does not share the common line with the pair. The Fourier transforms of the different side-views of the ATP synthase all share a single common line and the algorithm has no information that can be used to determine the orientations of the views about the long axis of the complex. Consequently, an initial model prepared by the common lines method could easily be based on incorrectly assigned projection angles, with the result that any signal deviating from the cylindrical average would be likely to be incorrect. With the current generation of projection matching EM structure refinement software, which tend to reinforce the initial model (Sigworth, 1998), it is important to begin structure refinement with a correct model. Rather than using a common lines method, we developed a method to test the hypothesis that the views represent a rotation series and, if verified, assign the relative orientation of each view about the axis. This method uses asymmetric features in the images as fiducials for determining the direction of projection. To emphasize the features in class-averages F–M that deviate from cylindrical symmetry, the average of all of the views was subtracted from each of the views. The remaining density, shown in Figure 3A, results from the asymmetric features in the structure. In these difference maps, this density can be seen as a continuous feature arising from the peripheral stalk of the complex. The feature starts at the top of F1 and ends as a domain in FO, and can even be seen in projections where it overlaps with the central stalk (Figure 3A, parts i, ii, v and vi). In Figure 3A, parts ii and vi, the feature decreases in intensity from its overlap with the negative signal created by the effect of the contrast transfer function of the microscope on the central stalk and edges of F1 and FO. Although the existence of a third stalk, that is a second peripheral stalk, has been proposed for the E.coli enzyme (Böttcher et al., 2000), from Figure 3A it is clear that there is only one peripheral stalk in the bovine mitochondrial enzyme. Figure 3.Rotation analysis. (A) Features that deviated from cylindrical symmetry in class-averages of Figure 2F–M were emphasized by subtracting the average of all of the views from each of the views. Markers were placed at maxima in the density to indicate the corresponding asymmetric features in each image. (B) To compare the experimental marker positions to the hypothesis that the images of the ATP synthase constitute a single-axis rotation series, the ratio of marker position to calculated marker radius (ai,j/ri) was plotted against the total angle for each marker in each image (φj + δi). The observations are consistent with the hypothesis that the series constitutes a rotation sequence, indicated by the dashed line (cos[φj + δi]). Download figure Download PowerPoint For a projection from a single-axis rotation series, the displacement, a, of a feature from the central vertical line of the image is given by: a = rfeaturecos(φprojection + δfeature) where rfeature is the radial distance between the feature and rotation axis, φprojection is the angle of projection about the rotation axis and δfeature is the relative angular offset of different features. As shown in Figure 3A, several markers were placed at maxima in the density at the same values of y in different images. The markers indicate the corresponding asymmetric features in the images. A conjugate gradient minimization method was used fit a value of φ for each image and r and δ for each marker. The output values of φ were 57, 38, 20, 274, 238, 210, 195 and 98° for images in Figure 2F–M. As an internal check on consistency, it can be seen that views that are approximate mirror images of each other have φ values that are ∼180° apart: 181° for F and J, 172° for G and K, 175° for H and L, and 176° for I and M. The asymmetric class-average from Figure 2P also conformed to the rotational model (at φ = 186°) and was therefore included in the analysis. It is possible that the 847 particle images contributing to class-averages L, P and M have been separated more successfully than the 896 images contributing to H and I. In Figure 3B, the ratio of ai,j/ri was plotted against (φj + δi) for each marker, where j denotes the image and i indicates the feature. Because each marker position was measured from the centre of each image, ai,j is the projection of a vector length ri on the x-axis and, if the set of images is a rotation series, the ratio ai,j/ri should be the cosine of the angle (φj + δi). A value on the x-axis of 0 or 360° indicates that a marker is at one extreme of its displacement from the central line of the image while 180° indicates that it is at the other extreme. The dashed line gives the hypothesis (cosine of the angle) that the images constitute a single-axis rotation series. From Figure 3B, it can be seen that the data are consistent with the rotation hypothesis (coefficient of determination, R2 = 0.990). Images in the rotational sequence were combined to make a ‘movie’ that gives the impression of the ATP synthase complex being rotated about its vertical axis (see Supplementary data available at The EMBO Journal Online or http://www2.mrc-lmb.cam.ac.uk/VA/Rubinstein/ATP_Synthase1.html). The ‘movie’ shows the two-domain structure of FO. It also reveals that the peripheral stalk is curved and wraps around the complex as it traverses the distance from the top of F1 to FO. It is apparent both from the rotational sequence and the difference maps in Figure 3 that in Figure 2F, G, J and K, the curved peripheral stalk is not missing, but is simply hidden by the central stalk. ATP_Synthase2.html shows the movement of marker positions used in the rotational analysis. The 3-D structure of the ATP synthase The φ angles obtained by fitting class-averages to the rotation hypothesis were used to generate a 3-D model of the ATP synthase (θ = 90°), which was then iteratively refined by projection matching. By the 0.143 threshold criteria for the Fourier shell correlation (Rosenthal et al., 2003), the resolution of the model was determined to be ∼32 Å. The peripheral stalk and the two domains of FO can be seen in surface rendered views of the EM map (Figure 4A and B). Since the shape of the density in the right-hand portion of Figure 4B strongly resembles the shape of the F1-c10 subcomplex determined by X-ray crystallography, we divided the experimental density into two segments using minima found in sections of the map to guide the partition. One segment consists of the peripheral stalk and second domain of FO and the other corresponds to the F1-c10 subcomplex (Figure 4C). In the model, the two domains of FO are of nearly equal volume. Other views of the partitioned model reveal the curvature of the peripheral stalk and a sharp bend near to its terminus at the central axis of F1 (Figure 5). Figure 4.3-D model of the ATP synthase. (A and B) Surface rendered views of the model after refinement. (C) The model after being divided into two parts. The first (blue) was chosen to correspond to the F1-c10 subcomplex and the remaining density (green) is interpreted to represent the peripheral stalk and second domain of FO. The grey mesh represents the experimental EM map. Download figure Download PowerPoint Figure 5.Two further views of the peripheral stalk. (A) A side-view of the model shows the curvature of the peripheral stalk. (B) A view from the F1 end of the assembly along its long axis reveals a sharp bend in the peripheral stalk near to its terminus. Download figure Download PowerPoint Several studies have observed the presence of a cap structure at the top of F1 (Wilkens and Capaldi., 1998b; Karrasch and Walker, 1999; Böttcher et al., 2000; Mellwig and Böttcher, 2003), although in one (Böttcher et al., 2000) the structure was reported to be evident only when the ATP synthase was inhibited with the non-hydrolysable ATP analogue AMP-PNP and not in the absence of this inhibitor. In these earlier studies, the cap appears as a structure sitting on top of F1 but disconnected from the peripheral stalk. In our model, the peripheral stalk is a continuous structure. However, since it is curved, there are several projections of the structure where the terminus is visible above F1, but the rest overlaps with F1 and the central stalk, giving the appearance of a cap. These views are seen in Figure 2F, G, J and K. As with any EM map built from untilted micrographs, the absolute hand of the map was initially unknown and arbitrarily chosen. Using a pair of tilted images recorded from the same field of particles and an automatic method (Rosenthal and Henderson, 2003), we tested the model to see if it is of the correct or incorrect hand. This procedure is known to be robust from its application to cases where the correct hand of the structure was already known. The plot in Figure 6 shows that the known magnitude and direction of the relative tilt between the pair of images can be determined independently by aligning particle images to the 3-D model. The location of the minimum indicates that the model is of the correct hand. The tilt experiment is also an entirely objective test of the structure: it provides further proof that the model is substantially correct and justifies the angular determination method described above. Figure 6.Determination of the absolute hand of the model. Tilt pairs of 29 different particles were recorded with a tilt between images of 30°. The first particle in each pair was aligned to the model. The plot shows the average phase residual between the model and the second particle in each pair for all possible tilt axes and tilts up to 40°. From the known direction and magnitude of the tilt between the images in the pair, a model of the correct hand was expected to produce a minimum at (2,−30°) as opposed to (−2,30°). The observed tilt axis and amount of tilt indicate that the model, as shown, is correct. Download figure Download PowerPoint Based on the crystal structure of the F1-c10 complex from S.cerevisiae (Stock et al., 1999) we assembled a model of F1-c10 consisting of the bovine F1-ATPase (Gibbons et al., 2000) and an oligomer of 10 E.coli c-subunits (Girvin et al., 1998; Stock et al., 1999). The number of c-subunits seems to vary in ATP synthase isolated from different organisms (Stock et al., 1999; Seelert et al., 2000; Stahlberg et al., 2001). The resolution of our EM data is insufficient to determine the number of c-subunits in the bovine enzyme. Consequently, the oligomer of 10 c-subunits was based on the stoichiometry observed in the yeast F1-c-ring assembly, the most intact complex for which a definitive structure is known. The F1-c10 structure was manually docked into the EM map (Figure 7). The EM map has several flattened sides that match the six flattened sides of the F1-ATPase crystal structure. These surfaces constrain the choice of orientation of F1-c10 to six positions in the EM map differing by 60° rotations about the long axis of the subcomplex (Figure 7B and C). The three catalytic α–β interfaces and the three non-catalytic interfaces create flattened sides with different dimensions. Although we do not think that it is possible to distinguish the α- from the β-subunits with complete certainty, the model shown gives the most likely orientation of the F1-c10 subcomplex within the map. It is apparent that along most of its contact region with F1, the peripheral stalk runs approximately along an α–β interface (red and yellow, respectively). In our docking, the peripheral stalk contacts a non-catalytic interface, leaving all three catalytic interfaces accessible. At the top of F1, the peripheral stalk is in contact almost entirely with an α-subunit. The modelled c10-ring appears to be smaller than the size of the c-ring domain in the EM map. This discrepancy may be the result of bound lipids that contribute to the size of the c-ring domain in the intact complex, a stoichiometry of more than 10 subunits in the bovine enzyme, or averaging of different conformations of the c-ring that leads to blurring and an exaggeration of its size in the EM map. Figure 7.Docking of an atomic model of F1-c10 into the EM map. The volume of the EM map not occupied by the atomic structure is coloured green. (A) A side-view of the model. The density that cannot be explained by the F1-c10 model consists of the peripheral stalk and the second domain of the FO region. This density must contain the remaining subunits of the ATP synthase: a, b, d, e, f, g, A6L, F6 and OSCP. (B–D) Cross-sections of the model. The terminus of the peripheral stalk at the top of F1 is on the central axis at the apex of the model (B). The peripheral stalk is primarily in contact with an α-subunit (red) near the top of F1, but is in contact with a non-catalytic interface between an α- (red) and β- (yellow) subunit along most of F1. The matching flattened sides of the F1 in the crystal structure and EM map show the uniqueness of fit between the two models. The central stalk and c-ring fit into the model well (D). Download figure Download PowerPoint Discussion The F1-c10 subcomplex contains 6 of the 16 different subunits of ATP synthase and accounts for 75% of its calculated mass. By dividing the model into the segment containing the known F1-c10 structure and the segment containing the other subunits, we found that the F1-c10 accounts for 78% of the model volume at the chosen threshold value. In the F1 region, the map agrees well with the atomic model, with the exception of the N termini of the α-subunits, which are known to interact with OSCP and ar