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4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virusThis study uses high-resolution cryo-electron microscopy to provide a complete structural model of the VEEV alphavirus, bridging the gap between incomplete crystal structures and lower resolution electron microscopy analyses.

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.

SUMMARY Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique which is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2-D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of ~3 compared to conventional cryoEM. The reconstruction was carried out to 13 Å resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and -cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.

SummaryCyanobacteria are photosynthetic organisms responsible for ~25% of organic carbon fixation on earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen billions of years ago. Cyanophages, which infect these bacteria, play an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike Phase Contrast (ZPC) electron cryo-tomography (cryoET) 1,2 . This imaging modality yields significant enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identified distinct Syn5 assembly Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms The averaged density maps of the procapsid, expanded capsid and the DNA-containing capsid have been deposited in the EBI under accession codes EMD-5742, EMD-5743, EMD-5744, EMD-5745, and EMD-5746, respectively. The authors declare no competing financial interests.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Fig. 1a-b) are roughly concentric, the thylakoid membrane does not fully enclose the inner compartment of the cell, nor does it seem to directly interact with the cell membrane. This differs from the organization seen in other cyanobacteria 9,10 . Cyanobacteria also contain carboxysomes, polyhedral compartments encapsulating enzymes for carbon fixation 11,12 . Each WH8109 cell has, on average, four or five carboxysomes, with diameters ranging from 920 to 1160Å (Fig. 1c). Ribosomes are abundant and widespread, forming numerous intracellular patches that contain polyribosomes (Fig. 1d). HHS Public AccessCyanophage Syn5 that infects WH8109 cells is a short-tailed icosahedral phage with a unique horn appendage at the vertex opposite to the tail 13 (Extended Data Fig. 2). Initial segmentation of our tomograms of infected cells identified Syn5 particles on the cell surface, floating in the extracellular medium, and Syn5 progeny inside the cell. Multiple full and empty phage particles are seen attached to the cell surface. Injection of viral DNA occurs at multiple sites on the bacterial envelope and does not appear to be a coordinated process. Fig. 1e shows a tubular density extending from the phage tail through the periplasm to the cytoplasm (Supplementary video 4), similar to observations in other phage-infected bacteria 14,15 . As infection progresses, increasing numbers of Syn5 phage progeny ...