Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

Cost-effective, efficacious therapeutics are urgently needed against the COVID-19 pandemic. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration and determined a structure of one of the most potent in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. We constructed multivalent Nb constructs that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization.

Summary Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3,290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687 and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), functional uniCOREs and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of new hypotheses.

Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical crosslinking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (ϳ600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope. Molecular & Cellular Proteomics

At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase leads the way in front to unwind DNA, and that DNA polymerases (Pol) trail behind the helicase. Here we use single particle electron microscopy to directly image a replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, while Ctf4 and the lagging strand Pol α-primase (Pol α) are behind the helicase. This unexpected architecture indicates that the leading strand DNA travels a long distance before reaching Pol ε, it first threads through the Mcm2-7 ring, then makes a U-turn at the bottom to reach Pol ε at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture, and provides a basis for further structural and biochemical replisome studies.

Summary The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14 MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and mRNP remodeling machinery right over the NPC's central channel, rather than on distal cytoplasmic filaments as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Despite the central role of large multi-protein complexes in many biological processes, it remains challenging to elucidate their structures and particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here, we present a strategy for isolating such complexes and for extracting distance restraints that allow the determination of their molecular architectures. The method was optimized to allow facile use of the extensive global resources of GFP-tagged transgenic cells and animals.

Transforming growth factor-␤-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor ␣ (TNF␣)-and interleukin-1␤ (IL-1␤)-induced IB kinase (IKK)/nuclear factor-B (NF-B) and c-Jun N-terminal kinase (JNK)/Tumor necrosis factor ␣ (TNF␣) 3 and interleukin-1␤ (IL-1␤) are two potent proinflammatory cytokines that play important roles in the regulation of immunity, inflammation, cell proliferation, differentiation, and apoptosis (1, 2). Cellular responses to TNF␣ and IL-1␤ are mediated by intracellular signaling pathways that control the activation of nuclear factor-B (NF-B) and activator protein 1 (AP-1) (3, 4).Upon binding to its receptor, TNF␣ induces formation of a receptor-associated complex, including the adaptor proteins TRADD, TRAF2, TRAF5, and RIP1, which subsequently leads to Lys 63 -linked polyubiquitination of TRAF2 and RIP1 (5-7). In contrast, IL-1␤ binding to its receptor induces a receptor-associated complex formation, including MyD88, IRAK1, IRAK4, and TRAF6, which is followed by Lys 63 -linked polyubiquitination of TRAF6 and IRAKs (8 -12). The formation of TRAF2-RIP1 and TRAF6-IRAK4 complexes as well as the Lys 63 -linked polyubiquitination of RIP1 and TRAF6 appear to enable the recruitment and activation of transforming growth factor-␤-activated kinase 1 (TAK1) through binding of the TAK1 regulatory subunits TAB2 and TAB3 to the Lys 63 -polyubiquitinated RIP1 and TRAF6. The activated TAK1 then triggers the activation of the IB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 MAPK (8,(13)(14)(15)(16)(17), which leads to activation of transcription factors NF-B and AP-1 and up-regulation of many genes encoding proinflammatory cytokines, chemokines, adhesion molecules, and proteolytic enzymes (18).The IKK complex consists of three subunits: two catalytic subunits, IKK␣ and IKK␤, and an essential regulatory subunit, IKK␥/NF-B essential modulator (NEMO) (3,19). Genetic studies have implicated that IKK␤ and IKK␥/NEMO are essential for the TNF␣-and IL-1␤-mediated . Phosphorylation of serine 177 and 181 residues in the activation loop is required for IKK␤ activation (23). IKK␥/NEMO has been indicated to bind Lys 63 -linked polyubiquitin chains (7,16,24,25). It is proposed that Lys 63 -polyubiquitin chains act as a scaffold to allow for assembly of a signaling complex that leads to IKK␤ activation. Once activated, IKK␤ phosphorylates IB proteins and leads to IB polyubiquitination with a Lys 48 -linked ubiquitin chain. Polyubiquitination-mediated degradation of IBs allows NF-B to translocate into the nucleus and activate NF-B-dependent gene expression (26).JNKs are members of three related mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs), JNKs, and p38 MAPKs (4, 27). JNKs and p38 MAPKs are involved in transmitting intracellular signals in