Image reconstruction from cryo-electron micrographs reveals the morphopoietic mechanism in the P2-P4 bacteriophage system.

Dokland, Terje; Lindqvist, Björn H.; Fuller, Stephen D.

doi:10.1002/j.1460-2075.1992.tb05121.x

Cited by 72 publications

(82 citation statements)

References 42 publications

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“…The most detailed structural information on dsDNA bacteriophage capsids has been determined by cryo-electron microscopy and image reconstruction (Conway et al, 1995;Dokland et al, 1992;Dokland & Murialdo, 1993;Prasad et al, 1993). Bacteriophage structure has also been investigated by X-ray solution scattering (King et al, 1976;Stroud et al, 1981) and spectroscopy (Li et al, 1981;Prevelige et al, 1993;Steven et al, 1990;Thomas et al, 1982).…”

Section: Introductionsupporting

confidence: 42%

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Wikoff

Duda

Hendrix

et al. 1999

Acta Crystallogr D Biol Cryst

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HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage !, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 A Ê in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 A Ê resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been con®rmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 A Ê . The cell dimensions after post-re®nement were a = 580, b = 626, c = 788 A Ê , = 90.0 . From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even re¯ections along the 0k0 lattice line indicate that the space group is P2 1 . The rotation function was used to determine the orientation of the particles in the unit cell and to con®rm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18 away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at ( 1 4 , 0, 1 4 ) and the other particle at ( 3 4 , 1 2 , 3 4 ). The particle position and orientation were con®rmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 A Ê is under way.

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Section: Introductionsupporting

confidence: 42%

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Wikoff

Duda

Hendrix

et al. 1999

Acta Crystallogr D Biol Cryst

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“…T ¼ 13 [1] and Q ¼ 20 [14] for T4. For most viruses, the T-number is a uniquely defined and evolutionarily robust parameter, although there are exceptions such as hepatitis B virus which produces capsids of both T ¼ 3 and T ¼ 4 [39], and the bacteriophage P2/P4 system which produces capsids of T ¼ 4 and T ¼ 7 [13].…”

Section: Polymorphism and Conformational Variabilitymentioning

confidence: 45%

Irregular and Semi‐Regular Polyhedral Models for Rous Sarcoma Virus Cores

Heymann

Butan

Winkler

et al. 2000

Computational and Mathematical Methods in Medicine

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Whereas many viruses have capsids of uniquely defined sizes that observe icosahedral symmetry, retrovirus capsids are highly polymorphic. Nevertheless, they may also be described as polyhedral foldings of a fullerene lattice on which the capsid protein (CA) is arrayed. Lacking the high order of symmetry that facilitates the reconstruction of icosahedral capsids from cryo-electron micrographs, the 3D structures of individual retrovirus capsids may be determined by cryoelectron tomography, albeit at lower resolution. Here, we describe computational and graphical methods used to construct polyhedral models that match in size and shape, capsids of Rous sarcoma virus (RSV) observed within intact virions. The capsids fall into several shape classes, including tubes, 'lozenges' and 'coffins'. The extent to which a capsid departs from icosahedral symmetry reflects the irregularity of the distribution of pentamers, which are always 12 in number for a closed polyhedral capsid. The number of geometrically distinct polyhedra grows rapidly with increasing quotas of hexamers, and ranks in the millions for particles in the size range of RSV capsids, which typically have 150-300 hexamers. Unlike the CAs of icosahedral viruses that assume a minimal number of quasi-equivalent conformations equal to the triangulation number (T), retroviral CAs exhibit a near-continuum of quasi-equivalent conformations -a property that may be attributed to the flexible hinge linking the N-and C-terminal domains.

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“…Vis-à-vis the P2 helper virus, Sid protein can be regarded as P2 assembly inhibitor. P2 mutations resistant to the actions of the Sid protein define a specific region of the P2 coat protein that can exclude the external scaffolding protein (7,17,22), much like the X174 mutations described here may exclude inhibitory D proteins from the D 3 positions.…”

Section: Discussionmentioning

confidence: 43%

Scaffolding Proteins Altered in the Ability To Perform a Conformational Switch Confer Dominant Lethal Assembly Defects

Cherwa

Uchiyama

Fane

2008

J Virol

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In the X174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D 1 D 2 and D 3 D 4 , in a non-quasi-equivalent structure. To achieve this arrangement, ␣-helix 3 assumes two different conformations: (i) kinked 30°at glycine residue 61 in subunits D 1 and D 3 and (ii) straight in subunits D 2 and D 4 . Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in ␣-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.

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Image reconstruction from cryo-electron micrographs reveals the morphopoietic mechanism in the P2-P4 bacteriophage system.

Cited by 72 publications

References 42 publications

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Irregular and Semi‐Regular Polyhedral Models for Rous Sarcoma Virus Cores

Scaffolding Proteins Altered in the Ability To Perform a Conformational Switch Confer Dominant Lethal Assembly Defects

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