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- EMDB-1825: Structural basis for scaffolding-mediated assembly and maturation... -

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Entry
Database: EMDB / ID: EMD-1825
TitleStructural basis for scaffolding-mediated assembly and maturation of a dsDNA virus
Map dataThis is the icosahedral density map for bacteriophage P22 empty procapsid solved by cryo-EM at 7.0-Angstrom resolution
Sample
  • Sample: Bacteriophage P22 empty procapsid
  • Virus: Enterobacteria phage P22 (virus)
Keywordsbacteriophage / phage / P22 / procapsid / icosahedral reconstruction / dsDNA virus
Biological speciesEnterobacteria phage P22 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsChen D-H / Baker ML / Hryc CF / DiMaio F / Jakana J / Wu W / Dougherty M / Haase-Pettingell C / Schmid MF / Jiang W ...Chen D-H / Baker ML / Hryc CF / DiMaio F / Jakana J / Wu W / Dougherty M / Haase-Pettingell C / Schmid MF / Jiang W / Baker D / King JA / Chiu W
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2011
Title: Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus.
Authors: Dong-Hua Chen / Matthew L Baker / Corey F Hryc / Frank DiMaio / Joanita Jakana / Weimin Wu / Matthew Dougherty / Cameron Haase-Pettingell / Michael F Schmid / Wen Jiang / David Baker / ...Authors: Dong-Hua Chen / Matthew L Baker / Corey F Hryc / Frank DiMaio / Joanita Jakana / Weimin Wu / Matthew Dougherty / Cameron Haase-Pettingell / Michael F Schmid / Wen Jiang / David Baker / Jonathan A King / Wah Chiu /
Abstract: 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 ...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 = 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.
#1: Journal: NAT.STRUCT.MOL.BIOL. / Year: 2003
Title: Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions
Authors: Jiang W / Li Z / Zhang Z / Baker ML / Prevelige PE Jr / Chiu W
History
DepositionNov 19, 2010-
Header (metadata) releaseDec 10, 2010-
Map releaseJan 28, 2011-
UpdateJan 28, 2011-
Current statusJan 28, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1825.map.gz / Format: CCP4 / Size: 300.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the icosahedral density map for bacteriophage P22 empty procapsid solved by cryo-EM at 7.0-Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 432 pix.
= 915.84 Å
2.12 Å/pix.
x 432 pix.
= 915.84 Å
2.12 Å/pix.
x 432 pix.
= 915.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.994226 - 1.82075
Average (Standard dev.)-0.000000000021863 (±0.115446)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-216-216-216
Dimensions432432432
Spacing432432432
CellA=B=C: 915.84 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z915.840915.840915.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-70-70-70
NX/NY/NZ140140140
MAP C/R/S123
start NC/NR/NS-216-216-216
NC/NR/NS432432432
D min/max/mean-0.9941.821-0.000

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Supplemental data

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Sample components

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Entire : Bacteriophage P22 empty procapsid

EntireName: Bacteriophage P22 empty procapsid
Components
  • Sample: Bacteriophage P22 empty procapsid
  • Virus: Enterobacteria phage P22 (virus)

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Supramolecule #1000: Bacteriophage P22 empty procapsid

SupramoleculeName: Bacteriophage P22 empty procapsid / type: sample / ID: 1000
Details: This sample is P22 empty procapsid from which the scaffolding proteins were removed by GuHCl.
Number unique components: 1

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Supramolecule #1: Enterobacteria phage P22

SupramoleculeName: Enterobacteria phage P22 / type: virus / ID: 1 / Name.synonym: P22 / NCBI-ID: 10754 / Sci species name: Enterobacteria phage P22 / Virus type: OTHER / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes / Syn species name: P22
Host (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
synonym: BACTERIA(EUBACTERIA)
Virus shellShell ID: 1 / Diameter: 610 Å / T number (triangulation number): 7

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.6 / Details: 50 mM Tris pH 7.6, 25 mM NaCl, 2mM EDTA
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4.2 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Top entry / Specimen holder model: JEOL
TemperatureMin: 4.2 K / Max: 4.2 K / Average: 4.2 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 400,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON COOLSCAN / Digitization - Sampling interval: 6.35 µm / Number real images: 570 / Average electron dose: 25 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each micrograph
Final angle assignmentDetails: EMAN:az, alt, phi
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.5 CUT-OFF
Software - Name: Multi-Path Simulated Annealing Optimization algorithm
Number images used: 18200
DetailsThe particles were selected using an automatic selection program

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