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- EMDB-1862: Visualization of a missing link in retrovirus capsid assembly -

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Basic information

Entry
Database: EMDB / ID: EMD-1862
TitleVisualization of a missing link in retrovirus capsid assembly
Map data
SampleIcosahedral assembly of Rous sarcoma virus capsid proteins:
Avian leukosis virus capsid protein
KeywordsRetrovirus / Capsid / Icosahedral
Biological speciesAvian leukosis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 10.4 Å
AuthorsCardone G / Purdy JG / Cheng N / Craven RC / Steven AC
CitationJournal: Nature / Year: 2009
Title: Visualization of a missing link in retrovirus capsid assembly.
Authors: Giovanni Cardone / John G Purdy / Naiqian Cheng / Rebecca C Craven / Alasdair C Steven /
Abstract: For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene ...For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJan 20, 2011-
Header (metadata) releaseFeb 4, 2011-
Map releaseFeb 4, 2011-
UpdateFeb 4, 2011-
Current statusFeb 4, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 297
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 297
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1862.map.gz / Format: CCP4 / Size: 30.9 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.27 Å/pix.
x 255 pix.
= 323.85 Å
1.27 Å/pix.
x 255 pix.
= 323.85 Å
1.27 Å/pix.
x 255 pix.
= 323.85 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.27 Å
Density
Contour LevelBy AUTHOR: 297.0 / Movie #1: 297
Minimum - Maximum-17762.0 - 15003.0
Average (Standard dev.)-5999.890000000000327 (±3146.550000000000182)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-127-127-127
Dimensions255255255
Spacing255255255
CellA=B=C: 323.85 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z1.271.271.27
M x/y/z255255255
origin x/y/z0.0000.0000.000
length x/y/z323.850323.850323.850
α/β/γ90.00090.00090.000
start NX/NY/NZ-160-160-159
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS-127-127-127
NC/NR/NS255255255
D min/max/mean-17762.00015003.000-5999.893

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Supplemental data

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Sample components

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Entire Icosahedral assembly of Rous sarcoma virus capsid proteins

EntireName: Icosahedral assembly of Rous sarcoma virus capsid proteins
Number of components: 1
Oligomeric State: 60 capsid protein subunits form 12 pentamers
MassTheoretical: 1.53 MDa

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Component #1: protein, Avian leukosis virus capsid protein

ProteinName: Avian leukosis virus capsid protein / a.k.a: RSV CA protein / Oligomeric Details: Icosahedral composed of 12 pentamers / Recombinant expression: Yes / Number of Copies: 60
MassTheoretical: 1.53 MDa
SourceSpecies: Avian leukosis virus / Strain: Prague C
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET-24

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL
Buffer solution: 10 mM Tris-HCl, 75 mM NaCl, 0.05 mM ETDA, 0.5 M NaPO4
pH: 7.5
Support filmHoley carbon film on 400 mesh gold grid
VitrificationInstrument: LEICA KF80 / Cryogen name: ETHANE / Temperature: 93.15 K
Method: 4.5 microliter sample dropped onto grid, blotted on one side for 1 second, then plunged
Details: Vitrification instrument: Reichert-Jung KF80 plunger. Vitrification carried out in nitrogen atmosphere

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 14 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 1500 nm
Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: 93.15 (88.15 - 98.15 K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 6 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 µm / Bit depth: 16 / OD range: 4.8

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 1478 / Applied symmetry: I (icosahedral)
3D reconstructionSoftware: EMAN, Bsoft, PFT2, EM3DR2
CTF correction: Each particle, phase reversal and baseline subtraction
Details: Particles were classified using EMAN and initial template generated using 3-fold class average. Preprocessing was done using Bsoft. For orientation assignment and 3D reconstruction PFT2 and ...Details: Particles were classified using EMAN and initial template generated using 3-fold class average. Preprocessing was done using Bsoft. For orientation assignment and 3D reconstruction PFT2 and EM3DR2 were used, repsectively
Resolution: 10.4 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: SITUS / Refinement protocol: rigid body / Target criteria: Laplacian correlation / Refinement space: RECIPROCAL
Details: Protocol: Rigid Body. The NTD domain was fitted automatically using colores
Input PDB model: 1EM9
Chain ID: 1EM9_A
Modeling #2Software: SITUS / Refinement protocol: rigid body / Target criteria: Laplacian correlation / Refinement space: RECIPROCAL
Details: Protocol: Rigid Body. The CTD domain (aa 152-230) was extracted and fitted using colacor. Model 14 was selected as the best fit.
Input PDB model: 1D1D
Chain ID: 1D1D_A

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