Using single particle electron cryomicroscopy that does not impose icosahedral averaging, we determined the structure of the entire infectious Salmonella phage Epsilon15 1 , including both icosahedral and non-icosahedral components. At least three layers of condensed viral DNA were observed to pack in coaxial coils with local 25 Å hexagonal inter-strand spacing. At one of the fivefold vertices, a portal complex with twelve subunits replaces a capsid pentamer. A tail hub with six projecting trimeric tailspikes sits on the external face of the portal. Below the portal is a cylindrical protein core. An extended shaft of density fills the central channel of the protein core and the portal complex and appears to consist of about 90 nucleotides at the terminus of the packaged DNA poised for injection. Using an icosahedral symmetry imposed reconstruction, the fold of the capsid shell protein is seen to resemble the capsid protein fold of other tailed double-stranded DNA phages 2-5 and human herpesvirus 6 . These common structural features suggest a common evolutionary origin among these viruses. Double-stranded DNA (dsDNA) phages are vectors for gene transfer among enteric bacteria, including important human pathogens 7 . For all the well-studied tailed dsDNA phages, a preformed procapsid shell is assembled, and the DNA is pumped into the shell through a portal complex located at a single vertex 8 . The phage tails are also assembled at this vertex. The portal complex together with packaging enzymes have been shown to function as components of a very powerful molecular motor 9 , but it has not been possible to visualize the complex within the intact virion.The inability to visualize the packed DNA and the portal vertex in the virion reflects the difficulties in determining these structural features which lack icosahedral symmetry and are lost in any icosahedral averaging used in X-ray crystallography or electron cryomicroscopy (cryoEM). Using a cryoEM single particle reconstruction technique without symmetry imposition, we have been able to determine the structure of these critical features of Salmonella phage Epsilon15, some of which are unexpected.Epsilon15 is a short tailed dsDNA bacteriophage that infects Salmonella anatum. Its genome (NCBI accession number: NC_004775) contains 39,671 base pairs with 49 open reading frames ( Supplementary Fig. 1) among which six, coding for structural proteins, were resolved by SDS-PAGE and identified by tryptic-digest/mass spectrometry ( Supplementary Fig. 2).

Summary Defects in primary cilia lead to devastating disease due to their roles in sensation and developmental signaling, but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain three-dimensional maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wildtype and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

The mechanisms by which most double-stranded DNA viruses package and release their genomic DNA are not fully understood. Single particle cryo-electron microscopy and asymmetric 3D reconstruction reveal the organization of the complete bacteriophage P22 virion, including the protein channel through which DNA is first packaged and later ejected. This channel is formed by a dodecamer of portal proteins and sealed by a tail hub consisting of two stacked barrels capped by a protein needle. Six trimeric tailspikes attached around this tail hub are kinked, suggesting a functional hinge that may be used to trigger DNA release. Inside the capsid, the portal's central channel is plugged by densities interpreted as pilot/injection proteins. A short rod-like density near these proteins may be the terminal segment of the dsDNA genome. The coaxially packed DNA genome is encapsidated by the icosahedral shell. This complete structure unifies various biochemical, genetic, and crystallographic data of its components from the past several decades.

Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214bp with a 237bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life-cycle. Assignment of eleven ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryoelectron micrographs of purified Syn5 virions revealed that the capsid has a single "horn", a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent three-fold rather than six-fold symmetry. An 18Å-resolution icosahedral reconstruction of the capsid revealed a T=7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria.

Herpes simplex virus type 1 is a human pathogen responsible for a range of illnesses from cold sores to encephalitis. The icosahedral capsid has a portal at one fivefold vertex which, by analogy to portal-containing phages, is believed to mediate genome entry and exit. We used electron cryotomography to determine the structure of capsids lacking pentons. The portal vertex appears different from pentons, being located partially inside the capsid shell, a position equivalent to that of bacteriophage portals. Such similarity in portal organization supports the idea of the evolutionary relatedness of these viruses.Herpes simplex virus type 1 (HSV-1) is a large virus with a diameter of Ͼ200 nm. The first step in herpesvirus particle assembly is the formation of an icosahedral capsid 125 nm in diameter (13). The viral genome is packaged into the capsid through a specialized structure known as the portal, which is located at one of the fivefold vertices (9, 15). The other 11 vertices are occupied by pentons. The portal is believed to be organized as a dodecameric ring similar to those found in tailed double-stranded DNA bacteriophages such as Epsilon15 and P22 (2, 3, 6). A single portal subunit has a mass of 74 kDa, which makes the entire portal ϳ888 kDa (9). This is similar to the mass of one penton (745 kDa) (16). This size equivalence at all vertices presents a challenge to efforts to determine the structure of the portal in the native capsid by any reconstruction method used in electron cryomicroscopy.To generate a mass difference between the vertex containing the portal and the other 11 vertices, we treated the capsids with 6 M urea. This has been shown to specifically remove pentons, peripentonal triplexes, small hexon-associated protein, protease, and scaffold from capsids without disrupting the hexontriplex shell (10). As shown in Fig. 1a, this treatment does not remove the portal protein (UL6), which is present in equivalent amounts in urea-treated and untreated capsids (11).The urea-extracted capsids were applied to Quantifoil grids and flash frozen in liquid ethane by use of a Vitrobot. The specimens were imaged at Ϫ180°C in a JEM2010F electron microscope at 200 kV on a Gatan 4kx4k charge-coupled device camera at ϫ55,360 effective magnification, 15 to 20 e/Å 2 dose, and 0.5 to 2.0 m defocus (Fig. 1b). Particle boxing and contrast transfer function parameters were determined with EMAN software (7). SAVR was used to reconstruct a threedimensional map with icosahedral symmetry imposed from randomly oriented capsid particles (4). This map shows holes at each of the fivefold vertices (Fig. 1c), confirming removal of most if not all of the pentons. The mass of the single portal vertex has been averaged over all 12 icosahedral fivefold positions, contributing insignificant density (nominally 1/12) to the reconstruction.In order to visualize the unique vertex which contains the portal, we used tomographic reconstruction, which does not impose any symmetry. The same urea-extracted preparation was mixed with 15-nm col...