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.