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- PDB-6ogz: In situ structure of Rotavirus RNA-dependent RNA polymerase at tr... -

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Basic information

Entry
Database: PDB / ID: 6ogz
TitleIn situ structure of Rotavirus RNA-dependent RNA polymerase at transcript-elongated state
Components
  • Inner capsid protein VP2
  • RNA (5'-R(P*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
  • RNA (5'-R(P*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
  • RNA-dependent RNA polymerase of rotavirus A
Keywordsviral protein/rna/transferase / RNA-dependent RNA polymerase / capsid shell protein / transcription / in situ structure / rotavirus / transcriptional factors / reovirus / VIRUS / viral protein-rna-transferase complex
Function / homology
Function and homology information


T=2 icosahedral viral capsid / viral inner capsid / viral genome replication / virion component / viral nucleocapsid / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding
Similarity search - Function
Viral RNA-directed RNA polymerase, 4-helical domain / Tetracycline Repressor; domain 2 - #80 / Four Helix Bundle (Hemerythrin (Met), subunit A) - #1390 / Four Helix Bundle (Hemerythrin (Met), subunit A) - #1400 / Ribosomal Protein S5; domain 2 - #140 / Alpha-Beta Plaits - #2480 / 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, 4-helical domain / Tetracycline Repressor; domain 2 - #80 / Four Helix Bundle (Hemerythrin (Met), subunit A) - #1390 / Four Helix Bundle (Hemerythrin (Met), subunit A) - #1400 / Ribosomal Protein S5; domain 2 - #140 / Alpha-Beta Plaits - #2480 / 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 / Rotavirus VP2 / Viral RNA-directed RNA-polymerase / Rotavirus VP2 protein / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Tetracycline Repressor; domain 2 / Ribosomal Protein S5; domain 2 / Four Helix Bundle (Hemerythrin (Met), subunit A) / Arc Repressor Mutant, subunit A / Alpha-Beta Plaits / DNA/RNA polymerase superfamily / Up-down Bundle / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / URIDINE 5'-TRIPHOSPHATE / RNA / RNA (> 10) / RNA-directed RNA polymerase / Inner capsid protein VP2
Similarity search - Component
Biological speciesRotavirus A
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDing, K. / Chang, T. / Shen, W. / Roy, P. / Zhou, Z.H.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DE025567 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)1S10RR23057 United States
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United 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.
History
DepositionApr 3, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2019Provider: repository / Type: Initial release
Revision 1.1May 29, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
B: RNA (5'-R(P*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
C: RNA (5'-R(P*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
A: RNA-dependent RNA polymerase of rotavirus A
E: Inner capsid protein VP2
F: Inner capsid protein VP2
G: Inner capsid protein VP2
I: Inner capsid protein VP2
H: Inner capsid protein VP2
J: Inner capsid protein VP2
K: Inner capsid protein VP2
L: Inner capsid protein VP2
M: Inner capsid protein VP2
N: Inner capsid protein VP2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,171,58415
Polymers1,170,57713
Non-polymers1,0072
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 2 types, 2 molecules BC

#1: RNA chain RNA (5'-R(P*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')


Mass: 5673.388 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Rotavirus A
#2: RNA chain RNA (5'-R(P*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')


Mass: 5367.222 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Rotavirus A

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Protein , 2 types, 11 molecules AEFGIHJKLMN

#3: Protein 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, RNA-directed RNA polymerase
#4: Protein
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

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE


Mass: 484.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Comment: UTP*YM
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM

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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-#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 FIELD / Cs: 2.7 mm
Image recordingElectron dose: 18 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32

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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: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 411438 / Symmetry type: POINT

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