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. 2012 Sep 11;109(37):14870-5.
doi: 10.1073/pnas.1213333109. Epub 2012 Aug 27.

Near-atomic resolution structural model of the yeast 26S proteasome

Florian Beck  1 , Pia Unverdorben, Stefan Bohn, Andreas Schweitzer, Günter Pfeifer, Eri Sakata, Stephan Nickell, Jürgen M Plitzko, Elizabeth Villa, Wolfgang Baumeister, Friedrich Förster
Affiliations

Near-atomic resolution structural model of the yeast 26S proteasome

Florian Beck et al. Proc Natl Acad Sci U S A. .

Abstract

The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
7.4 Å resolution EM single particle reconstruction of S.cerevisiae 26S proteasome without imposed symmetry. The density is displayed as an isosurface from two different views, on the right colored according to the local resolution as indicated by the color key. Five different slices from the upper RP (A–E) are shown and compared to the counterparts in the lower RP (A’–E’) by difference images.
Fig. 2.
Fig. 2.
Atomic model of the 26S holocomplex fitted into the cryo-EM map. (A) Holocomplex seen from three different views. In the model, the PCI subunits are colored in different shades of green (order from left to right in middle panel: Rpn9/5/6/7/3/12), the MPN subunits in light (Rpn8) and dark (Rpn11) magenta, the Ub receptors Rpn10 and Rpn13 in purple, Rpn1 in brown, Rpn2 in yellow, the AAA-ATPase hexamer in blue, and the CP in red. (B) Subunit models fitted into their corresponding experimental densities. The ubiquitin receptors Rpn10 and Rpn13 were omitted due to their small size and due to the low resolution of the EM map in the corresponding areas.
Fig. 3.
Fig. 3.
Structure of the AAA-ATPase hexamer. (A) The atomic model of the AAA-ATPase (dark blue: Rpt1/6/4; light blue: Rpt2/3/5) is displayed together with the segmented cryo-EM density in isosurface representation. The Walker A and Walker B motifs are colored in red and orange, respectively. The densities marked by the dashed ovals corresponds to the likely disordered N-termini. (B) Slice through the N-ring as indicated in A. (C) Slice through the AAA-fold.
Fig. 4.
Fig. 4.
Organization of Rpn8/Rpn11 dimer and lid C-termini. (A) Localization of the MPN domains (colored tan) and the coiled-coil bundle likely formed by the lid C-termini (green) in the context of the 26S holocomplex (red: CP, blue: AAA-ATPase hexamer, grey: remaining subunits) in a side view. (B) Same seen from top. (C) Magnified top-view of MPN densities and coiled-coil bundle. The atomic models of Rpn8 (light magenta) and Rpn11 (magenta) are fitted into the density. The interface of the MPN domains of Rpn8 and Rpn11 is formed by helices 8H1/11H1, 8H2/11H3, and 8H4/11H5. In the coiled-coil bundle Rpn11 helices 11H6 and 11H7 and Rpn8 helix 8H5 could be traced. (D) Rpn8/Rpn11 dimer in the context of AAA-ATPases and coiled-coil bundle.
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