Structure and assembly of the capsid of bacteriophage P22

King, Jonathan; Botstein, David; Casjens, Sherwood; Earnshaw, William C.; Harrison, S.C.; Lenk, Elaine V.

doi:10.1098/rstb.1976.0096

Cited by 62 publications

(44 citation statements)

References 21 publications

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“…The bacteriophage P22 procapsid is a well-characterized morphogenetic precursor of the mature phage, consisting of hundreds of copies of the gene 5 (gp5) coat and gene 8 (gp8) scaffolding proteins, multiple copies of three ejection proteins (gp7, gp16, gp20, also known as pilot proteins), and a unique multisubunit gene 1 (gp1) portal (13). We used single-particle cryo-EM to determine its structure at 3.8-Å resolution, enforcing icosahedral symmetry with approximately 23,400 particle images (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

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

202

319

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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...

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Section: Resultsmentioning

confidence: 99%

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

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

202

319

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“…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). Despite previous attempts, no high-resolution structures have been determined, primarily because crystals which diffracted X-rays could not be grown.…”

Section: Introductionmentioning

confidence: 99%

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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“…These efforts establish the groundwork for a quantitative assessment of a cryo-EM structure. The methods described here were applied to the capsid of P22 bacteriophage, which infects Salmonella and has been extensively studied through biochemistry, genetics, and biophysics (5)(6)(7)(8).…”

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confidence: 99%

Accurate model annotation of a near-atomic resolution cryo-EM map

Hryc

Chen

Afonine

et al. 2017

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

113

118

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Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.ecently, cryo-EM maps with associated atomic coordinates have been reported at resolutions better than 4 Å (1, 2). However, few have been subjected to rigorous evaluation of the reliability of the observed features or of the correlation between the experimental map and its corresponding model at the residue level. Typically, such correlation is reported in terms of a curve known as the Fourier shell correlation (FSC), which is a function of spatial frequency (3, 4). Although informative, it does not assure the authenticity of local features, nor does it indicate which features in the model agree or disagree with observed density. Ideally, a molecular model can be used to generate a map that replicates the experimental map in most or all of its details, and thus constitutes a trustworthy and informative model for the specimen's structure at the reported resolution. This study examines the agreement and/or disagreement between the model and the experimental map density, determined at nearatomic resolution. These efforts establish the groundwork for a quantitative assessment of a cryo-EM structure. The methods described here were applied to the capsid of P22 bacteriophage, which infects Salmonella and has been extensively studied through biochemistry, genetics, and biophysics (5-8). ResultsCryo-EM Images and Reconstructions. To study the P22 structure, we used a 300-keV electron cryomicroscope (JEM-3200FSC; JEOL Ltd.) and a Direct Electron detector (DE-20; operated in integrating mode) to collect frozen, hydrated P22 bacteriophage images (Fig. 1A and Table S1). Signal was detectable out to 3-Å resolution ( Fig. S1 and Movie S1). A total exposure of 37.5 e − /Å 2 was fractionated into 24 frames during a 1.5-s exposure. All frames were dose-weighted (4) and used to refine particle orientation parameters; empirically, we found that using image frames 1 to 6 (a cumulative exposu...

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Structure and assembly of the capsid of bacteriophage P22

Cited by 62 publications

References 21 publications

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Accurate model annotation of a near-atomic resolution cryo-EM map

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