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- EMDB-1710: Cryo-EM 3D model of the icosahedral particle composed of Rous sar... -

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Basic information

Entry
Database: EMDB / ID: EMD-1710
TitleCryo-EM 3D model of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers
Map data
SampleIcosahedral particles composed of Rous sarcoma virus capsid protein:
Capsid protein p27
Function / homology
Function and homology information


host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / structural constituent of virion / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / nucleic acid binding / aspartic-type endopeptidase activity / zinc ion binding
Aspartic peptidase, active site / Retroviral nucleocapsid protein Gag, p24 fragment / Retrovirus capsid, N-terminal / Retrovirus capsid, C-terminal / Retroviral Gag polyprotein, M / Peptidase A2A, retrovirus, catalytic / Retropepsins / Zinc finger, CCHC-type / Retroviral matrix protein / Matrix protein, lentiviral and alpha-retroviral, N-terminal ...Aspartic peptidase, active site / Retroviral nucleocapsid protein Gag, p24 fragment / Retrovirus capsid, N-terminal / Retrovirus capsid, C-terminal / Retroviral Gag polyprotein, M / Peptidase A2A, retrovirus, catalytic / Retropepsins / Zinc finger, CCHC-type / Retroviral matrix protein / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Aspartic peptidase domain superfamily / Retropepsin-like catalytic domain / Zinc finger, CCHC-type superfamily
Gag polyprotein
Biological speciesRous sarcoma virus - Prague C
Methodsingle particle reconstruction / cryo EM / Resolution: 18.3 Å
AuthorsHyun JK / Radjainia M / Kingston RL / Mitra AK
CitationJournal: J Biol Chem / Year: 2010
Title: Proton-driven assembly of the Rous Sarcoma virus capsid protein results in the formation of icosahedral particles.
Authors: Jae-Kyung Hyun / Mazdak Radjainia / Richard L Kingston / Alok K Mitra /
Abstract: In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell ...In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic resolution structural analysis. CA hexamers and pentamers are the fundamental building blocks of the capsid, however the pentameric state, in particular, remains poorly characterized. We have developed an efficient in vitro protocol for studying the assembly of Rous sarcoma virus (RSV) CA that involves mild acidification and produces structures modeling the authentic viral capsid. These structures include regular spherical particles with T = 1 icosahedral symmetry, built from CA pentamers alone. These particles were subject to cryoelectron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM-derived three-dimensional density map. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned underneath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra are the only detectable species and that these exist in reversible equilibrium at pH 5. These same acidic conditions are known to promote formation of a RSV CA CTD dimer, present within the icosahedral particle, which facilitates capsid assembly. The results are consistent with a model in which RSV CA assembly is a nucleation-limited process driven by very weak protein-protein interactions.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMar 11, 2010-
Header (metadata) releaseApr 9, 2010-
Map releaseApr 9, 2010-
UpdateApr 16, 2014-
Current statusApr 16, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 164.272806409
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 164.272806409
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2x8q
  • Surface level: 164.272806409
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2x8q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1710.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 160 pix.
= 400. Å
2.5 Å/pix.
x 160 pix.
= 400. Å
2.5 Å/pix.
x 160 pix.
= 400. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 162.0 / Movie #1: 164.2728064
Minimum - Maximum-397.634999999999991 - 686.375999999999976
Average (Standard dev.)5.10931 (±72.627799999999993)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 400 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z400.000400.000400.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-61-61-60
NX/NY/NZ122122122
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-397.635686.3765.109

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Supplemental data

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Sample components

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Entire Icosahedral particles composed of Rous sarcoma virus capsid protein

EntireName: Icosahedral particles composed of Rous sarcoma virus capsid protein
Details: The icosahedral particles were assembled in vitro, by transferring recombinant Rous sarcoma virus capsid protein monomers into high salt, mildly acidic buffer
Number of components: 1
Oligomeric State: Icosahedral particle containing 12 CA pentamers (i.e. 60 monomers)
MassTheoretical: 1.5 MDa

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Component #1: protein, Capsid protein p27

ProteinName: Capsid protein p27 / a.k.a: Capsid protein p27
Oligomeric Details: Icosahedral particle composed of 12 protein pentamers
Recombinant expression: Yes / Number of Copies: 60
MassTheoretical: 1.53 MDa
SourceSpecies: Rous sarcoma virus - Prague C
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.2 mg/mL
Buffer solution: 0.1M citric acid, 5mM MOPS/KOH, 725mM NaCl, 0.25mM Na azide, 0.125mM TCEP-HCl
pH: 5
Support filmHoley carbon 400 mesh copper grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 85 K / Humidity: 90 % / Method: Blot for 5 seconds before plunging / Details: Vitrification instrument: Vitrobot Mark IV

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 12
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 18 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 42000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 60,000 - 140,000 times magnification using live fft
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 3000 nm
Specimen HolderHolder: Side entry liquid nitrogen-cooled cryo specimen holder
Model: GATAN LIQUID NITROGEN / Temperature: 103
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 21 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 10.5 µm / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 1310 / Applied symmetry: I (icosahedral)
3D reconstructionAlgorithm: Polar Fourier transform / Software: Bsoft, PFT2, EM3DR2 / CTF correction: Each micrograph
Details: The digitized micrographs were processed using Bsoft. Orientation and origin search of the particles and 3D reconstruction were performed using PFT2 and EM3DR, respectively
Resolution: 18.3 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera, Sculptor / Refinement protocol: rigid body / Target criteria: Cross-correlation / Refinement space: RECIPROCAL
Details: Protocol: Rigid body. The domain structures were manually fitted into the 3D reconstruction, and then the fitting was refined using Sculptor
Input PDB model: 1EM9, 3G21
Output model

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