Structure of the Connector of Bacteriophage T7 at 8Å Resolution: Structural Homologies of a Basic Component of a DNA Translocating Machinery

Agirrezabala, Xabier; Martín‐Benito, Jaime; Valle, Mikel; González, José M.; Valencia, Alfonso; Valpuesta, José M.; Carrascosa, José L.

doi:10.1016/j.jmb.2005.02.005

Cited by 102 publications

(116 citation statements)

References 43 publications

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“…5a). This overall feature resembles that seen in other isolated portals with negligible sequence similarity [16][17][18][19] . The azimuthal density distribution of Epsilon15 portal deviates noticeably from perfect 12-fold symmetry (Fig.…”

supporting

confidence: 75%

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Jiang

Chang

Jakana³

et al. 2006

Nature

286

340

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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).

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supporting

confidence: 75%

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Jiang

Chang

Jakana³

et al. 2006

Nature

286

340

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“…The icosahedral shell was assigned to the only major capsid protein, gp10 (28). The 12-fold z5 region in FAR-I, FAR-II, FAR-IV, and FAR-V reconstructions was assigned to the portal protein, gp8, based on previous findings of both the same 12-fold symmetry in the structures of both purified portal proteins and portal proteins attached to tails ejected from phage (29,30). The densities of the 12-fold gp8 layer superimpose well on the X-ray diffraction-derived, 12-fold atomic model of SPP1 (31) and P22 (32) phage portal rings (Fig.…”

Section: Resultsmentioning

confidence: 99%

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7

Guo

Liu

Vago

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used singleparticle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.T he tailed dsDNA bacteriophages and human dsDNA viruses, such as herpesviruses and adenoviruses, share many features of structural organization and life cycle. Viruses of these diverse families evolved from a common ancestor that existed before the divergence of prokaryotes, archaea, and eukaryotes, the three domains of life (1-3). Assisted by a scaffolding protein, these viruses assemble a procapsid with a symmetrical, usually icosahedral, outer shell. One of the shell's 12 fivefold vertices is replaced by a symmetry-mismatched 12-fold portal. The dsDNA genome is subsequently pumped into the capsid chamber through the portal channel, accompanied by exit of scaffolding proteins. The energydependent DNA packaging process is performed by a complex machinery involving multiple, stacked layers at the portal vertex. Functions of the layers include DNA binding/loading (small terminase), ATPase hydrolysis (large terminase), DNA channel (portal), and DNA condensation (internal proteins, sometimes in several layers). The detailed structural basis and functional mechanism of the DNA packaging process are under extensive investigation (4-6).Structural analysis of phage portal vertex and associated proteins has impact beyond understanding viral assembly. Symmetry mismatches also occur in the capping and branching of actin filaments to control...

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“…Sample Preparation-Mature T7 virions were purified from E. coli lysates of BL21 cells infected with wild type phage using sucrose gradient centrifugation as described elsewhere (28,30). The composition of the purified viral particles was validated by SDS-PAGE analysis in the presence of ␤-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

“…Image acquisition and processing were performed as described previously (30). More specifically, the micrographs were recorded on Kodak SO-163 film under low dose conditions at a nominal magnification of 50,000ϫ, digitized in a Zeiss scanner (Photoscan TD, Z/I Imaging Corp.) using a 7-m step, and sampled to 0.14 nm/pixel.…”

Section: Methodsmentioning

confidence: 99%

Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7

Ionel

Velázquez-Muriel

Luque

et al. 2011

Journal of Biological Chemistry

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Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A-and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.

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Structure of the Connector of Bacteriophage T7 at 8Å Resolution: Structural Homologies of a Basic Component of a DNA Translocating Machinery

Cited by 102 publications

References 43 publications

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7

Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7

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