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- PDB-6ogy: In situ structure of Rotavirus RNA-dependent RNA polymerase at du... -

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Entry
Database: PDB / ID: 6ogy
TitleIn situ structure of Rotavirus RNA-dependent RNA polymerase at duplex-open state
Components
  • DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')
  • Inner capsid protein VP2
  • RNA (5'-R(P*AP*GP*CP*C)-3')
  • RNA-dependent RNA polymerase of rotavirus A
Keywordsviral protein/transferase/rna / RNA-dependent RNA polymerase / capsid shell protein / transcription / in situ structure / rotavirus / transcriptional factors / reovirus / VIRUS / viral protein-transferase-rna complex
Function / homology
Function and homology information


viral inner capsid / viral genome replication / virion / ec:2.7.7.48: / viral nucleocapsid / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / nucleotide binding / RNA binding
RNA-directed RNA polymerase, reovirus / Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Rotavirus VP1 C-terminal domain / Rotavirus VP2 protein / Viral RNA-directed RNA-polymerase / RNA-directed RNA polymerase, luteovirus / Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP2
RNA-directed RNA polymerase / Inner capsid protein VP2
Biological speciesRotavirus A
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDing, K. / Chang, T. / Shen, W. / Roy, P. / Zhou, Z.H.
Funding supportUnited States , 8件
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI094386United States
National Institutes of Health/National Institute of General Medical SciencesGM071940United States
National Institutes of Health/National Institute of Dental and Craniofacial ResearchDE025567United States
National Institutes of Health/National Center for Research Resources1S10RR23057United States
National Institutes of Health/Office of the Director1S10OD018111United States
National Institutes of Health/National Institute of General Medical SciencesU24GM116792United States
National Science Foundation (United States)DBI-1338135United States
National Science Foundation (United States)DMR-1548924United States
CitationJournal: Nat Commun / Year: 2019
Title: In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release.
Authors: Ke Ding / Cristina C Celma / Xing Zhang / Thomas Chang / Wesley Shen / Ivo Atanasov / Polly Roy / Z Hong Zhou /
Abstract: Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron ...Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 3, 2019 / Release: May 22, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 22, 2019Structure modelrepositoryInitial release
1.1May 29, 2019Structure modelData collection / Database referencescitation / citation_author_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: RNA-dependent RNA polymerase of rotavirus A
C: Inner capsid protein VP2
D: Inner capsid protein VP2
E: Inner capsid protein VP2
F: Inner capsid protein VP2
G: Inner capsid protein VP2
H: Inner capsid protein VP2
I: Inner capsid protein VP2
J: Inner capsid protein VP2
K: Inner capsid protein VP2
L: Inner capsid protein VP2
M: DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')
N: RNA (5'-R(P*AP*GP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)1,162,16713
Polymers1,162,16713
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide RNA-dependent RNA polymerase of rotavirus A / Protein VP1


Mass: 125276.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Rotavirus A / References: UniProt: G0YZJ9, EC: 2.7.7.48
#2: Protein/peptide
Inner capsid protein VP2 / Capsid shell protein of rotavirus A


Mass: 103425.992 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Rotavirus A / References: UniProt: G0YZK0
#3: DNA/RNA hybrid DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')


Mass: 1390.855 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Rotavirus A
#4: RNA chain RNA (5'-R(P*AP*GP*CP*C)-3')


Mass: 1239.818 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Rotavirus A

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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 A / Type: VIRUS / Entity ID: 1,2,3,4 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Rotavirus A
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingElectron dose: 22 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 25

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 798260 / Symmetry type: POINT

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