Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Summary Genome packaging into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike dsDNA viruses which pump their genome into a preformed capsid1-3, ssRNA viruses, such as bacteriophage MS2, co-assemble their capsid with genome4-7; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via host “sex” pilus (F-pilus)8 and is the first fully-sequenced organism9 and a model system for studies of gene translational regulations10,11, RNA-protein interactions12-14, and RNA virus assembly15-17. Its positive-sense ssRNA genome of 3569 bases is enclosed in a capsid with one maturation protein (MP) monomer and 89 coat protein (CP) dimers arranged in a T=3 icosahedral lattice18,19. MP is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8, but how the genome is organized and delivered are not known. Here we show the MS2 structure at 3.6Å resolution determined by electron-counting cryo electron microscopy (cryoEM) and asymmetric reconstruction. We traced ~80% backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-CP interactions among 15 of these stem-loops with diverse sequences. The stem-loop at 3’ end of the genome interacts extensively with the MP, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus, and joins 89 CP-dimers to form a capsid. This first atomic description of genome-capsid interactions in a spherical ssRNA virus provides insights into genome delivery via host “sex” pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires imaginations about links between nucleoprotein complexes and the origin of viruses.

The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR-Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon.

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require non-native constructs involving mutations or truncations, and are challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID , a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum , an organism that has resisted conventional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.

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Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an N-terminal foot-, a head-and body domains connected to an extended C-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C-terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc finger-like motifs are present in each NS1 monomer and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn 2+ , suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, while zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.