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- EMDB-3101: Cryo-electron tomography and subtomogram averaging of Rous-Sarcom... -

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Basic information

Entry
Database: EMDB / ID: EMD-3101
TitleCryo-electron tomography and subtomogram averaging of Rous-Sarcoma-Virus deltaMBD virus-like particles
Map dataSubtomogram averaging reconstruction of immature RSV capsid from virus-like particles
Sample
  • Sample: Immature-like Rous-Sarcoma Virus Gag particles
  • Protein or peptide: Rous-Sarcoma Virus deltaMBD Gag protein
KeywordsRetrovirus / 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 / viral translational frameshifting / host cell plasma membrane ...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 / viral translational frameshifting / 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 ...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 / Retroviral matrix protein / Retrovirus capsid, C-terminal / 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
Methodsubtomogram averaging / cryo EM / Resolution: 7.7 Å
AuthorsSchur FKM / Dick RA / Hagen WJH / Vogt VM / Briggs JAG
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 20, 2015-
Header (metadata) releaseAug 5, 2015-
Map releaseAug 12, 2015-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a9e
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5a9e
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3101.tif

Downloads & links

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Map

FileDownload / File: emd_3101.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram averaging reconstruction of immature RSV capsid from virus-like particles
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.07 Å/pix.
x 120 pix.
= 248.4 Å		2.07 Å/pix.
x 120 pix.
= 248.4 Å		2.07 Å/pix.
x 120 pix.
= 248.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-6.57726717 - 8.24688911
Average (Standard dev.)-0.00028003 (±0.7611295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 248.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.072.072.07
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z248.400248.400248.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-6.5778.247-0.000

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Supplemental data

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Sample components

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Entire : Immature-like Rous-Sarcoma Virus Gag particles

EntireName: Immature-like Rous-Sarcoma Virus Gag particles
Components
  • Sample: Immature-like Rous-Sarcoma Virus Gag particles
  • Protein or peptide: Rous-Sarcoma Virus deltaMBD Gag protein

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Supramolecule #1000: Immature-like Rous-Sarcoma Virus Gag particles

SupramoleculeName: 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

MacromoleculeName: Rous-Sarcoma Virus deltaMBD Gag protein / type: protein_or_peptide / ID: 1 / Name.synonym: RSV dMBD / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Rous sarcoma virus / synonym: RSV
Molecular weightTheoretical: 52 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceUniProtKB: Gag polyprotein

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 6.5 / Details: MES pH6.5, 100mM NaCl,2uM ZnCl2 2mM TCEP
GridDetails: C-Flat 2/2-#c grids, glow discharged for 30 sec in 20mA
VitrificationCryogen 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GATAN GIF 2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateSep 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 34 e/Å2 / Details: Tilt series consisted of 21 or 31 micrographs.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 45 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsReconstruction carried out using subtomogram averaging. Subtomogram averaging was performed using scripts from the TOM (Nickell et al, 2005), AV3 (Foerster et al, 2005) and Dynamo (Castano-Diez, 2012) packages. Subtomograms were extracted from the surface of the virus-like parcticles with the radius corresponding to the radius of the particles.
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.7 Å / Resolution method: OTHER / Software - Name: IMOD, AV3, TOM, Dynamo
Details: Reconstruction carried out using subtomogram averaging. Subtomogram averaging was performed using scripts from the TOM (Nickell et al, 2005), AV3 (Foerster et al, 2005) and Dynamo (Castano- ...Details: Reconstruction carried out using subtomogram averaging. Subtomogram averaging was performed using scripts from the TOM (Nickell et al, 2005), AV3 (Foerster et al, 2005) and Dynamo (Castano-Diez, 2012) packages. Subtomograms were extracted from the surface of the virus-like parcticles with the radius corresponding to the radius of the particles.
Number subtomograms used: 8375
CTF correctionDetails: Phase flipping of individual tilts
Final 3D classificationNumber classes: 1

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Atomic model buiding 1

Initial modelPDB ID:

1p7n

SoftwareName: Chimera, VMD, MDFF
DetailsStructures for p10 and CA-NTD (PDB 1P7N) and CA-CTD (PDB 3G1I, one monomer) were rigid body docked into the EM-density using the "Fit in map" option in chimera. Redundant residues of the antiparallel dimer of PDB 1P7N were removed. Missing residues in the helix7/helix8 linker region of CA, the connection loop between p10 and CA-NTD and residues upstream of the helix in p10 were manually modelled in Coot. The fit was further refined using Molecular Dynamics Flexible Fitting.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5a9e:
Cryo-electron tomography and subtomogram averaging of Rous-Sarcoma- Virus deltaMBD virus-like particles

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Atomic model buiding 2

Initial modelPDB ID:

3g1i

SoftwareName: Chimera, VMD, MDFF
DetailsStructures for p10 and CA-NTD (PDB 1P7N) and CA-CTD (PDB 3G1I, one monomer) were rigid body docked into the EM-density using the "Fit in map" option in chimera. Redundant residues of the antiparallel dimer of PDB 1P7N were removed. Missing residues in the helix7/helix8 linker region of CA, the connection loop between p10 and CA-NTD and residues upstream of the helix in p10 were manually modelled in Coot. The fit was further refined using Molecular Dynamics Flexible Fitting.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5a9e:
Cryo-electron tomography and subtomogram averaging of Rous-Sarcoma- Virus deltaMBD virus-like particles

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