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- EMDB-2100: Location of the dsRNA-dependent polymerase, VP1, in rotavirus par... -

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Basic information

Entry
Database: EMDB / ID: EMD-2100
TitleLocation of the dsRNA-dependent polymerase, VP1, in rotavirus particles
Map datareconstruction of rotavirus DLP+VP1 (polymerase)
Sample
  • Sample: Rotavirus DLP+VP1
  • Protein or peptide: Rotavirus polymerase (VP1)
  • Protein or peptide: VP1
  • Protein or peptide: VP2
  • Protein or peptide: VP3
  • RNA: dsRNARNA
Keywordsrotavirus / dsRNA-dependent / polymerase
Function / homology
Function and homology information


virion component => GO:0044423 / viral genome replication / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding
Similarity search - Function
Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP1 C-terminal domain / RNA-directed RNA polymerase, luteovirus / Viral RNA-directed RNA-polymerase / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase
Similarity search - Component
Biological speciesBovine rotavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsEstrozi LF / Settembre EC / Goret G / McClain B / Zhang X / Chen JZ / Grigorieff N / Harrison SC
CitationJournal: J Mol Biol / Year: 2013
Title: Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles.
Authors: Leandro F Estrozi / Ethan C Settembre / Gaël Goret / Brian McClain / Xing Zhang / James Z Chen / Nikolaus Grigorieff / Stephen C Harrison /
Abstract: Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a ...Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.
History
DepositionMay 14, 2012-
Header (metadata) releaseJun 14, 2012-
Map releaseJun 14, 2012-
UpdateJan 9, 2013-
Current statusJan 9, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0004844
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0004844
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4au6
  • Surface level: 0.0004844
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4au6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2100.png

Downloads & links

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Map

FileDownload / File: emd_2100.map.gz / Format: CCP4 / Size: 20.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of rotavirus DLP+VP1 (polymerase)
Voxel sizeX=Y=Z: 1.69643 Å
Density
Contour LevelBy AUTHOR: 0.0004844 / Movie #1: 0.0004844
Minimum - Maximum-0.00405112 - 0.00763549
Average (Standard dev.)0.00004119 (±0.00069179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1390-139
Dimensions279140140
Spacing279140140
CellA: 237.5 Å / B: 473.30356 Å / C: 237.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.69642857142861.69643010752691.6964285714286
M x/y/z140279140
origin x/y/z0.0000.0000.000
length x/y/z237.500473.304237.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS0-139-139
NC/NR/NS140279140
D min/max/mean-0.0040.0080.000

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Supplemental data

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Sample components

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Entire : Rotavirus DLP+VP1

EntireName: Rotavirus DLP+VP1
Components
  • Sample: Rotavirus DLP+VP1
  • Protein or peptide: Rotavirus polymerase (VP1)
  • Protein or peptide: VP1
  • Protein or peptide: VP2
  • Protein or peptide: VP3
  • RNA: dsRNARNA

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Supramolecule #1000: Rotavirus DLP+VP1

SupramoleculeName: Rotavirus DLP+VP1 / type: sample / ID: 1000
Oligomeric state: 780 molecules of VP6 form a DLP particle with 12 molecules of VP1, 120 molecules of VP2, 12 molecules of VP3 and 11 dsRNA molecules
Number unique components: 5

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Macromolecule #1: Rotavirus polymerase (VP1)

MacromoleculeName: Rotavirus polymerase (VP1) / type: protein_or_peptide / ID: 1 / Name.synonym: VP1
Details: The icosahedral 3D reconstruction of rotavirus DLP shows extra-density near the 5-fold axis corresponding to one copy of VP1 attached to the DLP inner surface.
Number of copies: 11 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus / synonym: Rotavirus
Molecular weightTheoretical: 126.326 KDa

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Macromolecule #2: VP1

MacromoleculeName: VP1 / type: protein_or_peptide / ID: 2 / Name.synonym: VP1 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus / synonym: Rotavirus

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Macromolecule #3: VP2

MacromoleculeName: VP2 / type: protein_or_peptide / ID: 3 / Name.synonym: VP2 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus / synonym: Rotavirus

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Macromolecule #4: VP3

MacromoleculeName: VP3 / type: protein_or_peptide / ID: 4 / Name.synonym: VP3 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus / synonym: Rotavirus

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Macromolecule #5: dsRNA

MacromoleculeName: dsRNA / type: rna / ID: 5 / Name.synonym: dsRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Bovine rotavirus / synonym: Rotavirus

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
GridDetails: Lacy carbon and C-flat
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Home-made. Vitrification carried out in air at room temperature
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 56540 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Eucentric, side-entry / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected
DateJun 1, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 386 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping for each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: RIco, and, FPM / Number images used: 7000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2r7o


Chain - Chain ID: A
SoftwareName: URO and VEDA
DetailsProtocol: Rigid body. The Fourier coefficients of VP1 were downweighted by a factor 5 in order to prevent VP1 from being "attracted" by the stronger density of the VP2/6 layer during the fit. matrix 1 0.286272 -0.958148 -0.000156 150.76188 0.573968 0.171358 0.800748 -71.02608 -0.767209 -0.229322 0.599001 -168.57402 matrix 2 -0.084128 -0.546853 -0.832991 184.93534 0.025364 -0.836859 0.546831 15.73129 -0.996132 0.024876 0.084274 -147.45338 matrix 3 0.245240 0.395934 -0.884926 125.30383 -0.558295 -0.688567 -0.462799 80.75011 -0.792568 0.607546 0.052184 -184.30794 matrix 4 0.819204 0.567290 -0.084182 54.27816 -0.370407 0.411304 -0.832843 34.17286 -0.437839 0.713450 0.547070 -228.20418 matrix 5 0.844563 -0.269594 0.462637 70.01368 0.329383 0.942768 -0.051920 -59.62970 -0.422162 0.196234 0.885026 -218.47982
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-4au6:
Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles

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