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Yorodumi- EMDB-3102: Cryo-electron tomography and subtomogram averaging of Rous-Sarcom... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3102 | |||||||||
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Title | Cryo-electron tomography and subtomogram averaging of Rous-Sarcoma-Virus deltaMBD virus-like particles | |||||||||
Map data | Tomogram containing one immature-like Rous-Sarcoma Virus Gag particle | |||||||||
Sample |
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Keywords | 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 / aspartic-type endopeptidase activity / nucleic acid binding / structural constituent of virion / host cell plasma membrane / proteolysis ...host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / aspartic-type endopeptidase activity / nucleic acid binding / structural constituent of virion / host cell plasma membrane / proteolysis / zinc ion binding / membrane Similarity search - Function | |||||||||
Biological species | Rous sarcoma virus | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Schur FKM / Dick RA / Hagen WJH / Vogt VM / Briggs JAG | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3102.map.gz | 10.9 MB | EMDB map data format | |
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Header (meta data) | emd-3102-v30.xml emd-3102.xml | 9.7 KB 9.7 KB | Display Display | EMDB header |
Images | emd_3102.tif | 1.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3102 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3102 | HTTPS FTP |
-Validation report
Summary document | emd_3102_validation.pdf.gz | 217.6 KB | Display | EMDB validaton report |
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Full document | emd_3102_full_validation.pdf.gz | 216.7 KB | Display | |
Data in XML | emd_3102_validation.xml.gz | 3.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3102 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3102 | HTTPS FTP |
-Related structure data
Related structure data | 3101C 5a9eC C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
EM raw data | EMPIAR-10037 (Title: Cryo-electron tomography and subtomogram averaging of Rous-Sarcoma-Virus deltaMBD virus-like particles Data size: 248.1 MB Data #1: Raw tilt series of a RSV dMBD virus-like particle [class averages]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3102.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Tomogram containing one immature-like Rous-Sarcoma Virus Gag particle | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.277 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Immature-like Rous-Sarcoma Virus Gag particles
Entire | Name: Immature-like Rous-Sarcoma Virus Gag particles |
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Components |
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-Supramolecule #1000: Immature-like Rous-Sarcoma Virus Gag particles
Supramolecule | Name: Immature-like Rous-Sarcoma Virus Gag particles / type: sample / ID: 1000 / Oligomeric state: Homohexameric / Number unique components: 1 |
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-Macromolecule #1: Rous-Sarcoma Virus deltaMBD Gag protein
Macromolecule | Name: Rous-Sarcoma Virus deltaMBD Gag protein / type: protein_or_peptide / ID: 1 / Name.synonym: RSV dMBD / Oligomeric state: Hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: Rous sarcoma virus / synonym: RSV |
Molecular weight | Theoretical: 52 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | UniProtKB: Gag polyprotein |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | particle |
-Sample preparation
Concentration | 5 mg/mL |
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Buffer | pH: 6.5 / Details: MES pH6.5, 100mM NaCl,2uM ZnCl2 2mM TCEP |
Grid | Details: C-Flat 2/2-#c grids, glow discharged for 30 sec in 20mA |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II Method: Degassed C-Flat 2/2-3C grids were glow discharged for 30 seconds at 20 mA. Virus solution was diluted in PBS containing 10nm colloidal gold. 2.5 ul of this mixture was applied to a grid. Blotting time: 2 seconds |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GATAN GIF 2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | Sep 10, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 34 e/Å2 / Details: Tilt series consisted of 21 or 31 micrographs. |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 45 ° / Tilt series - Axis1 - Angle increment: 3 ° |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | CTF correction was performed on each projection |
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Final reconstruction | Software - Name: IMOD Details: Deposited tomogram is binned 4x compared to the one used for final subtomogram averages. Number images used: 31 |
CTF correction | Details: Phase flipping of individual tilts |