Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method - PubMed (original) (raw)

Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method

Andres E Leschziner et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Chromatin remodeling complexes (remodelers) are large, multisubunit macromolecular assemblies that use ATP hydrolysis to alter the structure and positioning of nucleosomes. The mechanisms proposed for remodeler action on nucleosomes are diverse, and require structural evaluation and insights. Previous reconstructions of remodelers using electron microscopy revealed interesting features, but also significant discrepancies, prompting new approaches. Here, we use the orthogonal tilt reconstruction method, which is well suited for heterogeneous samples, to provide a reconstruction of the yeast RSC (remodel the structure of chromatin) complex. Two interesting features are revealed: first, we observe a deep central cavity within RSC, displaying a remarkable surface complementarity for the nucleosome. Second, we are able to visualize two distinct RSC conformers, revealing a major conformational change in a large protein "arm," which may shift to further envelop the nucleosome. We present a model of the RSC-nucleosome complex that rationalizes the single molecule results obtained by using optical tweezers and also discuss the mechanistic implications of our structures.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Analysis of RSC's amenability to the orthogonal tilt reconstruction method. Comparison of class averages obtained through reference-free alignment and classification of images collected at either 0° (horizontal rhomboid) or ±45° tilt (tilted rhomboid) showing the presence of similar characteristic views in both samples.

Fig. 2.

Fig. 2.

RSC conformational variability. (A) Reconstructions of two conformers of RSC. The initial models were obtained by using the Orthogonal Tilt Reconstruction method and then refined by projection-matching against 0° data. The names of the main features of the reconstructions are indicated in italics. The “open” and “closed” conformations differ mainly in the position of the arm (at the right of the structures shown in the top row). The bottom row shows the “top” and “back” views of the open conformer. A scale bar corresponding to 100 Å is shown. (B) Comparison of reprojections (“Prj”) of the structures shown in A with the best-matching reference-free class averages (“Avg”). (C) Four selected experimental class averages for RSC showing a variety of positions occupied by the arm (arrows).

Fig. 3.

Fig. 3.

Fitting of a nucleosome into the open and closed conformations of RSC. The crystal structure of the nucleosome (23) (PDB ID code 1AOI) was filtered to the resolution of the two reconstructions (37 Å) and manually fitted into each of the two reconstructed conformations. The filtered nucleosome is shown in yellow, and the RSC reconstructions are shown in gray.

Fig. 4.

Fig. 4.

Model of nucleosome binding by RSC. The x-ray crystal structure of the nucleosome (23) (PDB ID code 1AOI) was manually fitted into the central cavity of RSC. The nucleosome is shown as a ribbon diagram within a translucent surface representation filtered to 10 Å. The DNA is represented in gold, and the protein is represented in orange. Back (Left) and front (Right) views of the complex are shown. The entry/exit points of the nucleosomal DNA are indicated with green arrows, the dyad axis (blue cylinder) is indicated with a blue arrow, the histone H3 tail visible in the crystal structure is indicated with an orange arrow, and the binding site for the translocase domain is shown on the DNA with maroon arrows.

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