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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 dataThis is a 3D reconstruction of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers
Sample
  • Sample: Icosahedral particles composed of Rous sarcoma virus capsid protein
  • Protein or peptide: Capsid protein p27
Function / homology
Function and homology information


host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / structural constituent of virion / nucleic acid binding / aspartic-type endopeptidase activity / host cell plasma membrane / proteolysis ...host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / structural constituent of virion / nucleic acid binding / aspartic-type endopeptidase activity / host cell plasma membrane / proteolysis / zinc ion binding / membrane
Similarity search - Function
Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal ...Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily
Similarity search - Domain/homology
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.
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
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  • 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

map_EMD-1710...

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)
AnnotationThis is a 3D reconstruction of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers
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
Components
  • Sample: Icosahedral particles composed of Rous sarcoma virus capsid protein
  • Protein or peptide: Capsid protein p27

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Supramolecule #1000: Icosahedral particles composed of Rous sarcoma virus capsid protein

SupramoleculeName: Icosahedral particles composed of Rous sarcoma virus capsid protein
type: sample / ID: 1000
Details: The icosahedral particles were assembled in vitro, by transferring recombinant Rous sarcoma virus capsid protein monomers into high salt, mildly acidic buffer
Oligomeric state: Icosahedral particle containing 12 CA pentamers (i.e. 60 monomers)
Number unique components: 1
Molecular weightTheoretical: 1.5 MDa

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Macromolecule #1: Capsid protein p27

MacromoleculeName: Capsid protein p27 / type: protein_or_peptide / ID: 1 / Name.synonym: Capsid protein p27 / Number of copies: 60
Oligomeric state: Icosahedral particle composed of 12 protein pentamers
Recombinant expression: Yes
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 1.53 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration1.2 mg/mL
BufferpH: 5
Details: 0.1M citric acid, 5mM MOPS/KOH, 725mM NaCl, 0.25mM Na azide, 0.125mM TCEP-HCl
GridDetails: Holey carbon 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK IV / Details: Vitrification instrument: Vitrobot Mark IV / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 103 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 60,000 - 140,000 times magnification using live fft
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 10.5 µm / Number real images: 21 / Average electron dose: 18 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Bsoft, PFT2, EM3DR2
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
Number images used: 1310

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Atomic model buiding 1

Initial model(PDB ID:

1em9

,

3g21

)
SoftwareName: Chimera,Sculptor
DetailsProtocol: Rigid body. The domain structures were manually fitted into the 3D reconstruction, and then the fitting was refined using Sculptor
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-2x8q:
Cryo-EM 3D model of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers

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