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- PDB-2x8q: Cryo-EM 3D model of the icosahedral particle composed of Rous sar... -

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Basic information

Entry
Database: PDB / ID: 2x8q
TitleCryo-EM 3D model of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers
ComponentsCAPSID PROTEIN P27
KeywordsVIRUS / CAPSID PROTEIN / VIRAL MATRIX PROTEIN
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
gag protein p24 N-terminal domain / Retroviral Gag polyprotein, M / Zinc finger, CCHC-type superfamily / Retropepsin-like catalytic domain / Aspartic peptidase domain superfamily / Retropepsins / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / Retrovirus capsid, C-terminal ...gag protein p24 N-terminal domain / Retroviral Gag polyprotein, M / Zinc finger, CCHC-type superfamily / Retropepsin-like catalytic domain / Aspartic peptidase domain superfamily / Retropepsins / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / Retrovirus capsid, C-terminal / Peptidase A2A, retrovirus, catalytic / Aspartic peptidase, active site / Zinc finger, CCHC-type / Retroviral nucleocapsid protein Gag, p24 fragment
Gag polyprotein
Biological speciesROUS SARCOMA VIRUS - PRAGUE C
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 18.3 Å
Model type detailsCA ATOMS ONLY, CHAIN A
AuthorsK Hyun, J. / Radjainia, M. / Kingston, R.L. / Mitra, A.K.
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 Report
SummaryFull reportAbout validation report
History
DepositionMar 11, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 19, 2010Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-1710
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: CAPSID PROTEIN P27


Theoretical massNumber of molelcules
Total (without water)24,4721
Polymers24,4721
Non-polymers00
Water0
1
A: CAPSID PROTEIN P27
x 60


Theoretical massNumber of molelcules
Total (without water)1,468,33460
Polymers1,468,33460
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: CAPSID PROTEIN P27
x 5


  • icosahedral pentamer
  • 122 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)122,3615
Polymers122,3615
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: CAPSID PROTEIN P27
x 6


  • icosahedral 23 hexamer
  • 147 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)146,8336
Polymers146,8336
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein CAPSID PROTEIN P27 / / Coordinate model: Cα atoms only


Mass: 24472.230 Da / Num. of mol.: 1 / Fragment: RESIDUES 240-465
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ROUS SARCOMA VIRUS - PRAGUE C
Description: RECOMBINANT ROUS SARCOMA VIRUS CAPSID PROTEIN WAS PRODUCED BY HETEROLOGOUS EXPRESSION IN E. COLI AND PURIFIED TO HOMOGENIETY
Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): ROSETTA 2 / References: UniProt: P03322

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ICOSAHEDRAL PARTICLES COMPOSED OF ROUS SARCOMA VIRUS CAPSID PROTEIN
Type: VIRUS
Buffer solutionName: 0.1M CITRIC ACID, 5MM MOPS/KOH, 725MM NACL, 0.25MM NA AZIDE, 0.125MM TCEP-HCL
pH: 5
Details: 0.1M CITRIC ACID, 5MM MOPS/KOH, 725MM NACL, 0.25MM NA AZIDE, 0.125MM TCEP-HCL
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: CRYOGEN- ETHANE, HUMIDITY- 90, TEMPERATURE- 85, INSTRUMENT- VITROBOT MARK IV, METHOD- BLOT FOR 5 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 12
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 42000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2 mm
Specimen holderTemperature: 103 K
Image recordingElectron dose: 18 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 21
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Sculptormodel fitting
2UCSF Chimeramodel fitting
3Bsoft3D reconstruction
4EM3DR23D reconstruction
5PFT23D reconstruction
CTF correctionDetails: EACH MICROGRAPH
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: POLAR FOURIER TRANSFORM / Resolution: 18.3 Å / Num. of particles: 1310 / Nominal pixel size: 2.5 Å / Actual pixel size: 2.5 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1710.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model building
IDPDB-ID3D fitting-ID
11EM91
23G211
RefinementHighest resolution: 18.3 Å
Refinement stepCycle: LAST / Highest resolution: 18.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms223 0 0 0 223

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