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

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Basic information

Entry
Database: PDB / ID: 4au6
TitleLocation of the dsRNA-dependent polymerase, VP1, in rotavirus particles
ComponentsRNA-DEPENDENT RNA POLYMERASE
KeywordsVIRAL PROTEIN / DSRNA-DEPENDENT
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6 Å
AuthorsEstrozi, L.F. / Settembre, E.C. / Goret, G. / McClain, B. / Zhang, X. / Chen, J.Z. / Grigorieff, N. / Harrison, S.C.
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, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 13, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 31, 2012Group: Database references
Revision 1.2Jan 16, 2013Group: Database references
Revision 1.3Mar 20, 2013Group: Other
Revision 1.4Aug 30, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id

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Structure viewerMolecule:
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Assembly

Deposited unit
A: RNA-DEPENDENT RNA POLYMERASE
B: RNA-DEPENDENT RNA POLYMERASE
C: RNA-DEPENDENT RNA POLYMERASE
D: RNA-DEPENDENT RNA POLYMERASE
E: RNA-DEPENDENT RNA POLYMERASE


Theoretical massNumber of molelcules
Total (without water)631,6225
Polymers631,6225
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
RNA-DEPENDENT RNA POLYMERASE /


Mass: 126324.484 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) BOVINE ROTAVIRUS
References: UniProt: B3IWN8*PLUS, RNA-directed RNA polymerase
Sequence detailsTHIS SEQUENCE IS FITTED FROM SIMIAN ROTAVIRUS, UNIPROT ID 10922

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ROTAVIRUS DLP / Type: VIRUS
Buffer solutionpH: 7.4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30 / Date: Jun 1, 2007
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 56540 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1100 nm / Cs: 2 mm
Specimen holderTemperature: 90 K / Tilt angle max: 0.001 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 386
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UROmodel fitting
2VEDAmodel fitting
3FPM3D reconstruction
4RICO3D reconstruction
CTF correctionDetails: INDIVIDUAL PARTICLE PHASE FLIPPING
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: SYMMETRY-ADAPTED FUNCTIONS / Resolution: 6 Å / Num. of particles: 7000 / Nominal pixel size: 1.18 Å / Actual pixel size: 1.23 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2100.(DEPOSITION ID: 10810).
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: Cross-correlation coefficient / Details: METHOD--URO REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 2R7O

2r7o

RefinementHighest resolution: 6 Å
Refinement stepCycle: LAST / Highest resolution: 6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms43385 0 0 0 43385

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