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- PDB-5a9e: Cryo-electron tomography and subtomogram averaging of Rous-Sarcom... -

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Basic information

Entry
Database: PDB / ID: 5a9e
TitleCryo-electron tomography and subtomogram averaging of Rous-Sarcoma- Virus deltaMBD virus-like particles
ComponentsDELTAMBD GAG PROTEIN
KeywordsVIRAL PROTEIN / RETROVIRUS / ROUS-SARCOMA VIRUS / IMMATURE RETROVIRUS / VIRUS-LIKE-PARTICLE / CAPSID
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
MethodELECTRON MICROSCOPY / electron tomography / cryo EM / Resolution: 7.7 Å
AuthorsSchur, F.K.M. / Dick, R.A. / Hagen, W.J.H. / Vogt, V.M. / Briggs, J.A.G.
CitationJournal: J Virol / Year: 2015
Title: The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly.
Authors: Florian K M Schur / Robert A Dick / Wim J H Hagen / Volker M Vogt / John A G Briggs /
Abstract: The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) ...The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation.
IMPORTANCE: Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by ...IMPORTANCE: Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.
History
DepositionJul 21, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 12, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2015Group: Database references / Derived calculations / Other
Revision 1.2Aug 2, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Biological unit as software_defined_assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-3101
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  • Superimposition on EM map
  • EMDB-3101
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DELTAMBD GAG PROTEIN
B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN
F: DELTAMBD GAG PROTEIN
G: DELTAMBD GAG PROTEIN
H: DELTAMBD GAG PROTEIN
I: DELTAMBD GAG PROTEIN
J: DELTAMBD GAG PROTEIN
K: DELTAMBD GAG PROTEIN
L: DELTAMBD GAG PROTEIN
M: DELTAMBD GAG PROTEIN
N: DELTAMBD GAG PROTEIN
O: DELTAMBD GAG PROTEIN
P: DELTAMBD GAG PROTEIN
Q: DELTAMBD GAG PROTEIN
R: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)942,71918
Polymers942,71918
Non-polymers00
Water0
1
A: DELTAMBD GAG PROTEIN
B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN
F: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
2
K: DELTAMBD GAG PROTEIN
R: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
3
L: DELTAMBD GAG PROTEIN
M: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
4
G: DELTAMBD GAG PROTEIN
N: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
5
H: DELTAMBD GAG PROTEIN
O: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
6
I: DELTAMBD GAG PROTEIN
P: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
7
J: DELTAMBD GAG PROTEIN
Q: DELTAMBD GAG PROTEIN

B: DELTAMBD GAG PROTEIN
C: DELTAMBD GAG PROTEIN
D: DELTAMBD GAG PROTEIN
E: DELTAMBD GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)314,2406
Polymers314,2406
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1

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Components

#1: Protein
DELTAMBD GAG PROTEIN


Mass: 52373.262 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ROUS SARCOMA VIRUS / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P03322

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: electron tomography

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Sample preparation

ComponentName: IMMATURE ROUS-SARCOMA VIRUS GAG PARTICLES / Type: VIRUS
Buffer solutionName: MES PH6.5, 100MM NACL, 2UM ZNCL2, 2MM TCEP / pH: 6.5 / Details: MES PH6.5, 100MM NACL, 2UM ZNCL2, 2MM TCEP
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE
Details: DEGASSED C-FLAT 2 2-3C GRIDS WERE GLOW DISCHARGED FOR 30 SECONDS AT 20 MA. VIRUS SOLUTION WAS DILUTED IN OBS CONTAINING 10NM COLLOIDAL GOLD. 2.5UL OF THIS MIXTURE WAS APPLIED TO A GRID. ...Details: DEGASSED C-FLAT 2 2-3C GRIDS WERE GLOW DISCHARGED FOR 30 SECONDS AT 20 MA. VIRUS SOLUTION WAS DILUTED IN OBS CONTAINING 10NM COLLOIDAL GOLD. 2.5UL OF THIS MIXTURE WAS APPLIED TO A GRID. BLOTTING TIME 2.5 SECONDS.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Sep 10, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderTilt angle max: 45 ° / Tilt angle min: -45 °
Image recordingElectron dose: 34 e/Å2 / Film or detector model: GATAN MULTISCAN
Image scansNum. digital images: 31
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2UCSF Chimeramodel fitting
3VMDmodel fitting
4AV33D reconstruction
5Dynamo3D reconstruction
6IMOD3D reconstruction
7TOM Toolbox3D reconstruction
CTF correctionDetails: PHASE-FLIPPING OF INDIVIDUAL MICROGRAPHS
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionMethod: CROSS-CORRELATION / Resolution: 7.7 Å / Num. of particles: 8375 / Nominal pixel size: 2.07 Å / Actual pixel size: 2.07 Å
Details: RECONSTRUCTION CARRIED OUT USING SUBTOMOGRAM AVERAGING. SUBTOMOGRAM AVERAGING WAS PERFORMED USING SCRIPTS DERIVED FROM THE AV3 (FOERSTER, ET AL) TOM (NICKELL, ET AL) AND DYNAMO (CASTANO- ...Details: RECONSTRUCTION CARRIED OUT USING SUBTOMOGRAM AVERAGING. SUBTOMOGRAM AVERAGING WAS PERFORMED USING SCRIPTS DERIVED FROM THE AV3 (FOERSTER, ET AL) TOM (NICKELL, ET AL) AND DYNAMO (CASTANO-DIEZ, ET AL) PACKAGES. STRUCTURES FOR P10 AND CA-NTD (PDB 1P7N) AND CA-CTD (PDB 3G1I, ONE MONOMER) WERE RIGID BODY DOCKED INTO THE EM-MAP USING THE FIT-IN-MAP OPTION IN CHIMERA. REDUNDANT RESIDUES OF THE ANTIPARALLEL DIMER OF PDB 1P7N WERE REMOVED. MISSING RESIDUES IN THE HELIX 7-HELIX 8 LINKER REGION OF CA, THE CONNECTION LOOP BETWEEN P10 AND CA-NTD AND RESIDUES UPSTREAM OF THE HELIX IN P10 WERE MANUALLY MODELED USING COOT. THE FIT WAS FURTHER REFINED USING MOLECULAR DYNAMICS FLEXIBLE FITTING. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3101. (DEPOSITION ID: 13603).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--MOLECULAR DYNAMICS FLEXIBLE FITTING
RefinementHighest resolution: 7.7 Å
Refinement stepCycle: LAST / Highest resolution: 7.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17406 0 0 0 17406

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