|Entry||Database: EMDB / ID: EMD-1862|
|Title||Visualization of a missing link in retrovirus capsid assembly|
|Sample||Icosahedral assembly of Rous sarcoma virus capsid proteins:|
Avian leukosis virus capsid protein
|Keywords||Retrovirus / Capsid / Icosahedral|
|Biological species||Avian leukosis virus|
|Method||single particle reconstruction / cryo EM / Resolution: 10.4 Å|
|Authors||Cardone G / Purdy JG / Cheng N / Craven RC / Steven AC|
|Citation||Journal: 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 Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_1862.map.gz / Format: CCP4 / Size: 30.9 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.27 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Icosahedral assembly of Rous sarcoma virus capsid proteins
|Entire||Name: Icosahedral assembly of Rous sarcoma virus capsid proteins|
Number of components: 1
Oligomeric State: 60 capsid protein subunits form 12 pentamers
|Mass||Theoretical: 1.53 MDa|
-Component #1: protein, Avian leukosis virus capsid protein
|Protein||Name: 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|
|Mass||Theoretical: 1.53 MDa|
|Source||Species: Avian leukosis virus / Strain: Prague C|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Vector: pET-24|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 2 mg/mL|
Buffer solution: 10 mM Tris-HCl, 75 mM NaCl, 0.05 mM ETDA, 0.5 M NaPO4
|Support film||Holey carbon film on 400 mesh gold grid|
|Vitrification||Instrument: 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
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS CM200FEG|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 14 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 50000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 1500 nm|
|Specimen Holder||Holder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: 93.15 (88.15 - 98.15 K)|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 6 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 µm / Bit depth: 16 / OD range: 4.8|
|Processing||Method: single particle reconstruction / Number of projections: 1478 / Applied symmetry: I (icosahedral)|
|3D reconstruction||Software: 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
-Atomic model buiding
|Modeling #1||Software: 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 #2||Software: 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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