Supplementary Information. Structure of a RSC nucleosome Complex and Insights into Chromatin Remodeling
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1 Supplementary Information Structure of a RSC nucleosome Complex and Insights into Chromatin Remodeling Yuriy Chaban 1, Chukwudi Ezeokonkwo 1, Wen-Hsiang Chung 1, Fan Zhang 1, Roger D. Kornberg 2, Barbara Maier-Davis 2, Yahli Lorch 2, and Francisco J. Asturias 1* 1 Department of Cell Biology, The Scripps Research Institute North Torrey Pines Road, La Jolla CA Department of Structural Biology, Stanford University School of Medicine. Stanford CA *To whom correspondence should be addressed: asturias@scripps.edu - 1 -
2 SUPPLEMENTARY METHODS AND DISCUSSION EM data Analysis and evaluation Analysis of the RSC-nucleosome complex using negatively stained specimens RSC-nucleosome samples preserved in stain were prepared as described under Experimental Procedures, with flash-freezing replaced by staining with uranyl acetate. In parallel experiments, 0.5 mm ATP was added to the incubation buffer prior to negative staining. Random Conical Tilt (RCT) data collection and image analysis were carried out as described 1. About 3,000 particle images from samples with and without ATP were selected for final analysis and 3D reconstruction. The main difference between RSC-nucleosome volumes and a previously reported RCT reconstruction of RSC alone 1 was the presence of additional density in the central cavity, which appeared somewhat smaller than expected for a nucleosome. Although ATP has been reported to stabilize the RSC-nucleosome complex 2,3 comparison of the RSC-nucleosome reconstructions obtained from samples prepared with and without ATP did not reveal significant changes in the density in the central cavity, or a significant rearrangement of the RSC structure (Supplementary Fig. 1). Supplementary Figure 1. Reconstructions of the RSC nucleosome complex in the absence (left) and in the presence (right) of excess ATP. These reconstructions, calculated from images of particles preserved in stain, illustrate that addition of excess ATP does not result in major changes in the structure of the RSC complex. Data Processing and Calculation of a RSC cryo- EM reconstruction using a hybrid image analysis approach. An initial cryo-em RSC reconstruction generated by matching cryo images to projections of a RSC structure calculated using the Random Conical Tilt (RCT) method 4 from images of RSC particles preserved in stain 1 seemed to represent a significant improvement over the initial stain reconstruction of RSC (Supplementary Fig. 2) but showed a significant amount of noise and failed to improve in resolution as refinement of image alignment parameters progressed. Reference-free alignment within groups of cryo-images aligned to projections of the RCT reconstruction revealed that images related to the front view of RSC prevalent in stained samples were properly aligned, but images corresponding to other views showed significant misalignment. This seemed understandable due to the almost complete lack of features in the front and back of the initial RCT reconstruction that resulted from particle deformation in stain and the absence of perpendicular projections in stained RSC samples. Supplementary Figure 2: Initial cryo-em RSC reconstruction obtained by using as a reference a RSC volume calculated using the random conical tilt method and images of RSC particles preserved in stain. The volume was filtered to 25 Å and flood-filled. The structure shows the central cavity observed in the stain reconstruction, but appears significantly wider in right and left views. Therefore, a hybrid image alignment strategy was pursued, in which images corresponding to projections of the reference structure not significantly affected by stain-induced deformation were separated by supervised classification, and misaligned images were subject to reference-free - 2 -
3 alignment and unsupervised classification 5. At the end of each classification step the homogeneity of each class average was tested by comparing the outcomes of repeated (usually 10) rounds of reference-free classification, and the relative angular orientation of averages arising from homogenous groups was determined using a modified version of the common lines algorithm implemented in SPIDER 6, in which the Euler angles from some projections were kept fixed, while the rest were allowed to vary (C. Yang, unpublished). To summarize, this hybrid analysis approach could be described as a process in which a missing cone of information is reduced by progressive addition of new projections (Supplementary Supplementary Figure 3: Hybrid image analysis protocol in which alternative rounds of reference-based and reference free alignment and classification are used in combination with a modified common lines procedure to gradually fill-up a cone of missing information. Fig. 3). Consecutive iterations of this procedure resulted in progressive improvement of the cryo-em structure as described below. The 11,974 RSC cryo-images were CTF corrected by phase flipping and treated as a single group. They were subjected to reference-based alignment using the initial cryo-em RSC structure (Supplementary Fig. 2) as a reference. RSC images were separated into groups corresponding to 83 reference projections (with consecutive references separated by 15 ). The outcomes from repeated rounds of reference-free alignment within each of these groups were compared to assess the homogeneity of each group, and 45 out of the 83 initial groups were selected. Euler angles for projections related to the front view of the RSC structure were assigned based on values from the corresponding RSC structure re-projections and kept constant (which stabilizes the outcome of the procedure), while the relative orientation of other class averages was determined using the modified common lines protocol. The resulting structure was then used as reference to run 8 rounds of refinement by projection matching 7 of the entire image data set using coarsely (10-15 ) spaced reference projections. The entire procedure was repeated, and averages from 55 homogeneous classes were used to generate an improved RSC volume Supplementary Figure 4: (a) RSC structure obtained after two cycles of hybrid image classification and alignment, as described in the text. Densities extending from the structure in the left and right views appeared increasingly pronounced. (b) Improved RSC structure obtained after refinement by projection matching of the cryo-em data set, using as reference the final volume obtained the hybrid image classification and alignment strategy
4 (Supplementary Fig. 4a). This improved RSC reconstruction was used as reference for 8 cycles of refinement by projection matching of the cryo-em data set (appropriately divided into defocus groups), and resulted in a significantly improved cryo-em RSC reconstruction (Supplementary Fig. 4b). This reconstruction was used as the initial reference for iterative refinement of the full RSC data set (~27,000 images) yielding the final RSC reconstruction presented in Figure 1 and the Supplementary Movie 1. Evaluation of the final cryo-em RSC reconstruction revealed a fairly isotropic distribution of RSC views and a resolution (estimated using the Fourier Shell Correlation criterion) of ~25 Å (Supplementary Fig. 5a). Further evaluation of the final cryo-em RSC reconstruction revealed a close correspondence between projections of the reconstruction and reference-free class averages of the aligned data (Supplementary Fig. 5b). Conformational changes in bottom domain of the RSC complex. RSC projections corresponding to views related to the front view of the complex (domain overlap obscured the bottom domain in other views) were sorted into groups using supervised classification. Pixels under a mask including the bottom domain were subjected to correspondence analysis and classification as described in Experimental Procedures. Class averages showed the bottom domain in a range of conformations that varied from closed to widely open. Consideration of the extreme closed and open conformations illustrates the Supplementary Figure 5: Evaluation of the RSC cryo-em reconstruction. (a) Fourier shell correlation plot used to estimate the resolution of the RSC reconstruction at ~25 Å (left), and angular distribution of projections in the RSC cryo-em data set (right). The position of a circle in the angular distribution plot relates to the orientation of the images it represents and its diameter is proportional to the number of images assigned to that particular view. (b) Re-projections of the RSC reconstruction (top half of each row) compared to reference-free class averages calculated from particles assigned to each specific view (bottom half of each row). The agreement between re-projections and unsupervised averages for different views indicates that images were adequately separated into homogenous groups and supports the conclusion that the reconstruction correctly represents the RSC structure. range of motion of the bottom domain (Supplementary Movie 2). These observations imply that the bulky appearance of the bottom domain in our RSC and RSC-nucleosome 3D reconstructions is at least partially related to comparatively lower resolution of that portion of the reconstructions due to high mobility. Our analysis also suggests that only the tip of the bottom domain is involved in a swinging motion that leads to the opening or closing of the central cavity (partial rotation of the domain - 4 -
5 would also be consistent with our observations). Further experiments will be required to provide a precise description of movement of the bottom, the factors that control it, and its possible functional significance. Evaluation of RSC-nucleosome complex stability and RSC-nucleosome images The stability of the RSC-nucleosome complex was evaluated under the precise conditions used for EM sample preparation (Supplementary Fig. 6a). Binding of the nucleosome to RSC was also tested by careful classification of RSC-nucleosome EM images (Supplementary Fig. 6b-d). Supplementary Figure 6: Stability and 2D EM analysis of the RSC-nucleosome complex. (a) Assessment of RSC-nucleosome binding by gel electrophoresis. The K d for formation of the RSC-nucleosome complex was estimated from the slope of a double reciprocal plot of the intensity of the free nucleosome band in the gel as a function of RSC concentration. [nuc] is the change in free nucleosome concentration, and [RSC] is the change in free RSC concentration. The K d for nucleosome RSC interaction was determined from the slope of the curve (slope = K d / starting nucleosome concentration). (b) A mixed RSC/RSC-nucleosome data set composed of image related to front views of the RSC complex were subjected to correspondence analysis focused on the pixels comprising the central cavity and immediately adjacent area. The histogram on the left shows the distribution of eigenvalue averages for six representative views (error bars indicate the standard deviation). The inset in the left panel depicts eigenimages corresponding to the first and second eigenvalues for the same six groups, showing that image variability is related to the presence or absence of density in the cavity. (c) Plotting the distribution of images with respect to the first two eigenvectors shows a single population of empty particles for the RSC data, but a bimodal distribution of RSC-nucleosome images, with a majority of them being classified as having density in the cavity (right panel). (d) 2D class averages for several views - 5 -
6 related to the front view of the RSC complex showing particles with and without density in the central cavity (top two rows), and difference between them showing the shape of the additional density in the cavity (bottom row). Analysis of the nucleosome density in the RSC-nucleosome reconstruction. The density apparent in the central cavity of the RSC-nucleosome reconstruction was analyzed by comparing it to a low-resolution (25 Å) model generated from the X-ray structure of the nucleosome 8. This comparison and the resulting docking of the histone core can be better appreciated in an animated movie showing the structures from different angles (Supplementary Movie 4). In the model the histones are shown in space-filling representation; H2A yellow, H2B red, H3 blue, H4 green. One of the H2A/H2B dimers was excluded from the model as the corresponding density was only partially resolved because the dimer was closely associated with the mobile bottom RSC domain and could not be segmented from RSC at the limited resolution of the cryo-em reconstruction. Furthermore, the fit of the histones in the central density is not perfect, leaving open the possibility that interaction with RSC might induce changes in the structure of the octamer. SUPPLEMENTARY MOVIE LEGENDS Supplementary Movie 1. Cryo-EM reconstruction of the RSC chromatin remodeling complex. The movie starts by showing a front view of RSC. Supplementary Movie 2. RSC bottom domain movement viewed from the position close to the front view. Each RSC image corresponds to a different RSC view. Supplementary Movie 3. Cryo-EM reconstruction of the RSC-nucleosome complex and comparison with the reconstruction of RSC alone. The RSC reconstruction is shown in yellow and the RSCnucleosome reconstruction as a combination of blue mesh (majority of the RSC-nucleosome volume) and solid blue (additional density resulting from nucleosome binding and revealed by subtracting the RSC reconstruction from the RSC-nucleosome reconstruction). The movie starts by showing a front view of the RSC and RSC-nucleosome volumes. Supplementary Movie 4. Docking of a model of the histones into the central portion of the density apparent in the RSC-nucleosome reconstruction. The movie starts from a top view of the EM density. The histones then appear and the EM density and histone model rotate together. At the end of the movie the RSC reconstruction is displayed to illustrate the fit of the histones in the central RSC cavity
7 SUPPLEMENTARY REFERENCES 1. Asturias, F.J., Chung, W.H., Kornberg, R.D. & Lorch, Y. Structural analysis of the RSC chromatin-remodeling complex. Proc Natl Acad Sci U S A 99, (2002). 2. Lorch, Y., Cairns, B.R., Zhang, M. & Kornberg, R.D. Activated RSC-nucleosome complex and persistently altered form of the nucleosome. Cell 94, (1998). 3. Saha, A., Wittmeyer, J. & Cairns, B.R. Chromatin remodeling through directional DNA translocation from an internal nucleosomal site. Nat Struct Mol Biol 12, (2005). 4. Radermacher, M. Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J Electron Microsc Tech 9, (1988). 5. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies, 342 (Academic Press, San Diego, 1996). 6. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116, (1996). 7. Penczek, P., Grassucci, R.A. & Frank, J. The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53, (1994). 8. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. Crystal structure of the nucleosome core particle at 2.8Å resolution. Nature 389, (1997)
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