The 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAAATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.coiled coils | mass spectrometry | proteasome-COP9-eIF3 domain | proteasome-cyclosome repeats I n eukaryotes, the ubiquitin-proteasome pathway (UPP) is essential for proteostasis: Misfolded proteins or otherwise defective proteins as well as short-lived regulatory proteins are eliminated by degradation (1). The UPP regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (for review see ref.2). The 26S proteasome is the most downstream element of the UPP, executing the degradation of polyubiquitylated substrates (3-5). It consists of the barrelshaped core particle (CP; approximately 700 kDa), which sequesters the proteolytically active site in its central cavity, and the regulatory particle (RP; approximately 900 kDa), which is attached at either one or both ends of the CP and prepares substrates for degradation (6).The RP consists of 19 different canonical subunits, including six regulatory particle AAA-ATPase subunits (Rpt1-6) and 13 regulatory particle non-ATPase subunits (Rpn1-3, Rpn5-13, and Rpn15). The integral ubiquitin (Ub) receptors Rpn10 and Rpn13 recognize polyubiquitylated substrates (7-9). Alternatively, polyubiquitylated substrates can be recruited by shuttling Ub-receptors (Dsk2, Rad23, Ddi2), which bind to substrates with their Ub-associated domain, and to Rpn1, Rpn10, or Rpn13 at their Ub-like domain (5). The metalloprotease Rpn11 deubiquitylates substrates prior to their degradation (10, 11). The functions of the other Rpn subunits remain to be established. The AAA-ATPases form a hexameric ring that unfolds substrates, opens the gate to the CP, and eventually translocates the substrates to the CP.Electron microscopy (EM) (12) and X-...

The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed the native structure and organization of the cytoplasmic translation machinery. Analysis of a large dynamic structure-the nuclear pore complex-revealed variations detectable at the level of individual complexes. Cryo-ET was used to visualize previously elusive structures, such as nucleosome chains and the filaments of the nuclear lamina, in situ. Elucidation of the lamina structure provides insight into its contribution to metazoan nuclear stiffness.

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/transporter (CP/T) was variable in volume and could occupy different positions along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold.

The mass spectrometric identification of chemically cross-linked peptides (CXMS) specifies spatial restraints of protein complexes; these values complement data obtained from common structure-determination techniques. Generic methods for determining false discovery rates of cross-linked peptide assignments are currently lacking, thus making data sets from CXMS studies inherently incomparable. Here we describe an automated target-decoy strategy and the software tool xProphet, which solve this problem for large multicomponent protein complexes.