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- PDB-6ric: Structure of the core Vaccinia Virus DNA-dependent RNA polymerase... -

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Basic information

Entry
Database: PDB / ID: 6ric
TitleStructure of the core Vaccinia Virus DNA-dependent RNA polymerase complex
Components
  • (DNA-dependent RNA polymerase subunit ...RNA polymerase) x 4
  • (DNA-directed RNA polymerase ...Polymerase) x 4
  • RAP94 transcription factor
KeywordsVIRAL PROTEIN / Vaccinia / RNA polymerase / Transcription / Gene expression
Function / homology
Function and homology information


virion component => GO:0044423 / viral transcription / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-binding transcription factor activity / DNA-templated transcription / DNA binding / zinc ion binding
Similarity search - Function
RNA polymerase-associated transcription specificity factor Rap94 / RNA polymerase-associated transcription specificity factor, Rap94 / DNA-directed RNA polymerase, 18kDa subunit, poxviral / DNA-directed RNA polymerase, 35kDa subunit, poxviral / RNA polymerase, 22kDa subunit, poxviral / DNA-directed RNA polymerase, 19kDa subunit, poxviral / DNA-directed RNA polymerase, 7kDa polypeptide, chordopoxviral / RNA polymerase, 30kDa subunit, chordopox-type / RNA polymerase, 132kDa subunit, poxvirus-type / RNA polymerase, 30kDa subunit, chordopox-type, N-terminal ...RNA polymerase-associated transcription specificity factor Rap94 / RNA polymerase-associated transcription specificity factor, Rap94 / DNA-directed RNA polymerase, 18kDa subunit, poxviral / DNA-directed RNA polymerase, 35kDa subunit, poxviral / RNA polymerase, 22kDa subunit, poxviral / DNA-directed RNA polymerase, 19kDa subunit, poxviral / DNA-directed RNA polymerase, 7kDa polypeptide, chordopoxviral / RNA polymerase, 30kDa subunit, chordopox-type / RNA polymerase, 132kDa subunit, poxvirus-type / RNA polymerase, 30kDa subunit, chordopox-type, N-terminal / Poxvirus DNA-directed RNA polymerase, 18 kD subunit / Poxvirus DNA-directed RNA polymerase, 35 kD subunit / Poxvirus RNA polymerase 22 kDa subunit / Poxvirus DNA-directed RNA polymerase 19 kDa subunit / Chordopoxvirus DNA-directed RNA polymerase 7 kDa polypeptide (RPO7) / Poxvirus DNA dependent RNA polymerase 30kDa subunit / Poxvirus DNA dependent RNA polymerase / Zinc finger TFIIS-type signature. / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 7 kDa subunit / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase 147 kDa polypeptide / Protein H4 / DNA-directed RNA polymerase 18 kDa subunit / DNA-directed RNA polymerase 19 kDa subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase 35 kDa subunit
Similarity search - Component
Biological speciesVaccinia virus GLV-1h68
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsGrimm, C. / Hillen, H.S. / Bedenk, K. / Bartuli, J. / Neyer, S. / Zhang, Q. / Huettenhofer, A. / Erlacher, M. / Dienemann, C. / Schlosser, A. ...Grimm, C. / Hillen, H.S. / Bedenk, K. / Bartuli, J. / Neyer, S. / Zhang, Q. / Huettenhofer, A. / Erlacher, M. / Dienemann, C. / Schlosser, A. / Urlaub, H. / Boettcher, B. / Szalay, A. / Cramer, P. / Fischer, U.
Funding support Germany, 7items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)SPP1935 Germany
German Federal Ministry for Education and ResearchEXC 2067/1- 390729940 Germany
European Research Council (ERC)TRANSREGULON Germany
Volkswagen Foundation Germany
German Research Foundation (DFG)Fi 573 7-2 Germany
German Research Foundation (DFG)Fi 573 18-1 Germany
CitationJournal: Cell / Year: 2019
Title: Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes.
Authors: Hauke S Hillen / Julia Bartuli / Clemens Grimm / Christian Dienemann / Kristina Bedenk / Aladar A Szalay / Utz Fischer / Patrick Cramer /
Abstract: Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate mG-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of ...Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate mG-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.
History
DepositionApr 23, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: DNA-dependent RNA polymerase subunit rpo147
B: DNA-dependent RNA polymerase subunit rpo132
C: DNA-directed RNA polymerase 35 kDa subunit
E: DNA-directed RNA polymerase subunit
F: DNA-directed RNA polymerase 19 kDa subunit
G: DNA-dependent RNA polymerase subunit rpo18
I: RAP94 transcription factor
J: DNA-dependent RNA polymerase subunit rpo7
S: DNA-directed RNA polymerase 30 kDa polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)505,34414
Polymers505,0589
Non-polymers2865
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area67980 Å2
ΔGint-348 kcal/mol
Surface area133040 Å2
MethodPISA

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Components

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DNA-dependent RNA polymerase subunit ... , 4 types, 4 molecules ABGJ

#1: Protein DNA-dependent RNA polymerase subunit rpo147 / RNA polymerase


Mass: 146995.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL125 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1I2, DNA-directed RNA polymerase
#2: Protein DNA-dependent RNA polymerase subunit rpo132 / RNA polymerase


Mass: 133526.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL194 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1Q1, DNA-directed RNA polymerase
#6: Protein DNA-dependent RNA polymerase subunit rpo18 / RNA polymerase


Mass: 17917.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL147 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1K4, DNA-directed RNA polymerase
#8: Protein DNA-dependent RNA polymerase subunit rpo7 / RNA polymerase


Mass: 7299.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL107 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1G3, DNA-directed RNA polymerase

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DNA-directed RNA polymerase ... , 4 types, 4 molecules CEFS

#3: Protein DNA-directed RNA polymerase 35 kDa subunit


Mass: 35430.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL205 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1R2, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit / Polymerase


Mass: 21365.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL123 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1I0, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase 19 kDa subunit


Mass: 19020.088 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL167 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1M4, DNA-directed RNA polymerase
#9: Protein DNA-directed RNA polymerase 30 kDa polypeptide


Mass: 29834.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL076 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1D1, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules I

#7: Protein RAP94 transcription factor


Mass: 93667.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL130 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1I7

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Non-polymers , 2 types, 5 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Core Vaccinia DNA-dependent RNA polymerase complexCOMPLEX#1-#90MULTIPLE SOURCES
2DNA-dependent RNA polymerase subunitsRNA polymeraseCOMPLEX#1-#91NATURAL
Molecular weightValue: 0.5 MDa / Experimental value: YES
Source (natural)Organism: Vaccinia virus GLV-1h68
Source (recombinant)Organism: Vaccinia virus
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.41 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3609
Image scansMovie frames/image: 39

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3CTF correction
5WarpCTF correction
8Cootmodel fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 479618
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152591 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00630269
ELECTRON MICROSCOPYf_angle_d0.73940906
ELECTRON MICROSCOPYf_dihedral_angle_d11.28218402
ELECTRON MICROSCOPYf_chiral_restr0.0534643
ELECTRON MICROSCOPYf_plane_restr0.0065165

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