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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 dataIcosahedral assembly of Rous sarcoma virus capsid proteins
Sample
  • Sample: Icosahedral assembly of Rous sarcoma virus capsid proteins
  • Protein or peptide: 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.
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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1862.png

emd_1862.png

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)
AnnotationIcosahedral assembly of Rous sarcoma virus capsid proteins
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
Components
  • Sample: Icosahedral assembly of Rous sarcoma virus capsid proteins
  • Protein or peptide: Avian leukosis virus capsid protein

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Supramolecule #1000: Icosahedral assembly of Rous sarcoma virus capsid proteins

SupramoleculeName: Icosahedral assembly of Rous sarcoma virus capsid proteins
type: sample / ID: 1000
Oligomeric state: 60 capsid protein subunits form 12 pentamers
Number unique components: 1
Molecular weightTheoretical: 1.53 MDa

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Macromolecule #1: Avian leukosis virus capsid protein

MacromoleculeName: Avian leukosis virus capsid protein / type: protein_or_peptide / ID: 1 / Name.synonym: RSV CA protein / Number of copies: 60 / Oligomeric state: Icosahedral composed of 12 pentamers / Recombinant expression: Yes
Source (natural)Organism: Avian leukosis virus / Strain: Prague C
Molecular weightTheoretical: 1.53 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-24

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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

Concentration2 mg/mL
BufferpH: 7.5
Details: 10 mM Tris-HCl, 75 mM NaCl, 0.05 mM ETDA, 0.5 M NaPO4
GridDetails: Holey carbon film on 400 mesh gold grid
VitrificationCryogen name: ETHANE / Chamber temperature: 93.15 K / Instrument: LEICA KF80
Details: Vitrification instrument: Reichert-Jung KF80 plunger. Vitrification carried out in nitrogen atmosphere
Method: 4.5 microliter sample dropped onto grid, blotted on one side for 1 second, then plunged

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 88.15 K / Max: 98.15 K / Average: 93.15 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 6 / Average electron dose: 14 e/Å2 / Od range: 4.8 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each particle, phase reversal and baseline subtraction
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 10.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Bsoft, PFT2, EM3DR2
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
Number images used: 1478

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Atomic model buiding 1

Initial modelPDB ID:

1em9


Chain - Chain ID: A
SoftwareName: SITUS
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. The NTD domain was fitted automatically using colores
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Laplacian correlation

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Atomic model buiding 2

Initial modelPDB ID:

1d1d


Chain - Chain ID: A
SoftwareName: SITUS
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. The CTD domain (aa 152-230) was extracted and fitted using colacor. Model 14 was selected as the best fit.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Laplacian correlation

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