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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 | ||||||
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Title | Cryo-EM 3D model of the icosahedral particle composed of Rous sarcoma virus capsid protein pentamers | ||||||
![]() | CAPSID PROTEIN P27![]() | ||||||
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Function / homology | ![]() host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / structural constituent of virion / ![]() ![]() ![]() | ||||||
Biological species | ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
Model type details | CA ATOMS ONLY, CHAIN A | ||||||
![]() | K Hyun, J. / Radjainia, M. / Kingston, R.L. / Mitra, A.K. | ||||||
![]() | ![]() 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 | ![]() ![]() ![]() | ||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmcif format | ![]() ![]() ![]() |
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-Related structure data
Related structure data | ![]() 1710CM C: citing same article ( M: map data used to model this data |
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Similar-shape strucutres |
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Links
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Assembly
Deposited unit | ![]()
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1 | ![]()
| x 60
2 |
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3 | ![]()
| x 5
4 | ![]()
| x 6
5 | ![]()
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Symmetry | Point symmetry: (Schoenflies symbol![]() ![]() |
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Components
#1: Protein | ![]() Mass: 24472.230 Da / Num. of mol.: 1 / Fragment: RESIDUES 240-465 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Description: RECOMBINANT ROUS SARCOMA VIRUS CAPSID PROTEIN WAS PRODUCED BY HETEROLOGOUS EXPRESSION IN E. COLI AND PURIFIED TO HOMOGENIETY Production host: ![]() ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: ICOSAHEDRAL PARTICLES COMPOSED OF ROUS SARCOMA VIRUS CAPSID PROTEIN Type: VIRUS |
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Buffer solution | Name: 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 |
Specimen | Conc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Specimen support | Details: HOLEY CARBON |
Vitrification![]() | Instrument: 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
Microscopy | Model: FEI TECNAI 12 |
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Electron gun | Electron source![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Temperature: 103 K |
Image recording | Electron dose: 18 e/Å2 / Film or detector model: KODAK SO-163 FILM |
Image scans | Num. digital images: 21 |
Radiation wavelength | Relative weight: 1 |
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Processing
EM software |
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CTF correction![]() | Details: EACH MICROGRAPH | ||||||||||||||||||||||||
Symmetry | Point symmetry![]() ![]() | ||||||||||||||||||||||||
3D reconstruction![]() | Method: 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 building | Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY | ||||||||||||||||||||||||
Atomic model building |
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Refinement | Highest resolution: 18.3 Å | ||||||||||||||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 18.3 Å
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