Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8-and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T ¼ 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.sDNA viruses infecting both prokaryotes and eukaryotes share a common assembly pathway proceeding from a precursor (procapsid) to an infectious virion (1-4). In addition to the coat proteins, the procapsid requires scaffolding proteins, absent from the virion, for proper assembly, and a portal for DNA packaging and subsequent DNA ejection. However, despite a half-century of research on icosahedral viruses, it remains unclear how initially identical subunits adopt both hexameric and pentameric conformations in the virus and select the correct locations needed to form closed shells of the proper size (5). Packaging of DNA through the portal is accompanied by the exit of scaffolding proteins from the procapsid and conformational changes in the coat proteins as the capsid matures (2, 6).Understanding the molecular mechanisms of dsDNA virus assembly and maturation requires knowledge of the interactions among the coat, scaffolding, and portal proteins, all of which are essential for these processes. X-ray crystallography (7-9) and electron cryomicroscopy (cryo-EM) (10-12) have yielded nearatomic to atomic resolution models of several dsDNA icosahedral viruses and provided a structural framework of interactions among their coat proteins. However, the structural details of procapsid portal incorporation, scaffolding protein bind...

Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, ~1MDa, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates1,2,3. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid4,5, which is triggered by ATP hydrolysis6. The structural rearrangements and molecular events leading to lid closure are still unknown. Here, we report four single particle cryo-EM structures of Mm-cpn, an archaeal group II chaperonin5,7, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 Å resolution of the closed conformation allowed building of the first ever atomic model directly from the cryo-EM density map, in which we were able to visualize the nucleotide and over 70% of the sidechains. The model of the open conformation was obtained by using the deformable elastic network modeling with the 8 Å resolution open state cryo-EM density restraints. Together, the open and closed structures reveal how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analysis reveals an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signaling driving the conformational cycle of group II chaperonins. Beyond this, we anticipate our methodology of combining single particle cryo-EM and computational modeling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative alpha helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.

Cryo-electron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Data Bank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB) and the National Center for Macromolecular Imaging (NCMI), is a global ‘one-stop shop’ resource for deposition and retrieval of cryoEM maps, models and associated metadata. The resource unifies public access to the two major archives containing EM-based structural data: EM Data Bank (EMDB) and Protein Data Bank (PDB), and facilitates use of EM structural data of macromolecules and macromolecular complexes by the wider scientific community.

Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single particle cryo-electron microscopy (cryo-EM) yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6 Å and 9 Å resolution, respectively. The asymmetric reconstruction reveals how symmetry mismatches are accommodated among 5 of the gene products at the portal vertex. Reconstructions of infectious and empty particles show a conformational change of the “valve” density in the nozzle, an orientation difference in the tail fibers, a disordering of the C-terminus of the portal protein, and disappearance of the core proteins. In addition, cryo-electron tomography (cryo-ET) of P-SSP7 infecting Prochlorococcus demonstrated the same tail fiber conformation as in empty particles. Our observations suggest a mechanism whereby, upon binding to the host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex that triggers DNA release.

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis. We found that the germline cyst in Xenopus contains 16 cells that are similar in general architecture and molecular composition to the cyst in Drosophila. Nest cells are connected by cytoplasmic bridges that contain ring canal-like structures. The nest cells contain a structure similar to the Drosophila fusome that that is probably involved in anchoring of the centrioles and organization of the primary mitochondrial cloud (PMC) around the centriole. We also find that in contrast to other organisms, in Xenopus, apoptosis is a rare event within the developing ovary. Our studies indicate that the processes responsible for the formation of female germline cysts and the establishment of germ cell polarity are highly conserved between invertebrates and vertebrates. The dissimilarities between Drosophila and Xenopus and the uniqueness of each system probably evolved through modifications of the same fundamental design of the germline cyst.

Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycleChaperonins are multisubunit entities that are composed of two stacked rings enclosing a central chamber for ATP-dependent protein folding. A series of cryo-EM structures of the eukaryotic group II chaperonin TRiC/CCT reveal the conformational changes during the ATPase cycle and provide insight into how the subunits cooperate to close the lid.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.