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- PDB-6rid: Structure of Vaccinia Virus DNA-dependent RNA polymerase elongati... -

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Basic information

Entry
Database: PDB / ID: 6rid
TitleStructure of Vaccinia Virus DNA-dependent RNA polymerase elongation complex
Components
  • (DNA-dependent RNA polymerase subunit ...RNA polymerase) x 4
  • (DNA-directed RNA polymerase ...Polymerase) x 4
  • Non-template strand DNA
  • RNA
  • Template strand DNA
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-templated transcription / DNA binding / zinc ion binding
Similarity search - Function
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 ...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 / DNA (> 10) / RNA / RNA (> 10) / 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 / DNA-directed RNA polymerase 18 kDa subunit / DNA-directed RNA polymerase 19 kDa subunit ...DNA / DNA (> 10) / RNA / RNA (> 10) / 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 / 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
Vaccinia virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHillen, H.S. / Bartuli, J. / Grimm, C. / Dienemann, C. / Bedenk, K. / Szalar, A. / Fischer, U. / Cramer, P.
Funding support Germany, 7items
OrganizationGrant numberCountry
German Research FoundationSFB860 Germany
German Research FoundationSPP1935 Germany
German Federal Ministry for Education and ResearchEXC 2067/1- 390729940 Germany
European Research CouncilTRANSREGULON Germany
Volkswagen Foundation Germany
German Research FoundationFi 573 7-2 Germany
German Research FoundationFi 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 / _citation_author.identifier_ORCID / _citation_author.name

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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
J: DNA-dependent RNA polymerase subunit rpo7
S: DNA-directed RNA polymerase 30 kDa polypeptide
N: Non-template strand DNA
T: Template strand DNA
P: RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)450,98116
Polymers450,69511
Non-polymers2865
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area56570 Å2
ΔGint-303 kcal/mol
Surface area128230 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
#7: 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
#8: 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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DNA chain , 2 types, 2 molecules NT

#9: DNA chain Non-template strand DNA


Mass: 14775.473 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus
#10: DNA chain Template strand DNA


Mass: 14786.517 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus

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RNA chain , 1 types, 1 molecules P

#11: RNA chain RNA /


Mass: 9742.849 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus

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Non-polymers , 2 types, 5 molecules

#12: Chemical ChemComp-MG / MAGNESIUM ION


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


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

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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
1Vaccinia Virus DNA-dependent RNA polymerase elongation complexCOMPLEX#1-#110MULTIPLE SOURCES
2DNA-directed RNA polymerase subunitsPolymeraseCOMPLEX#1, #3-#81RECOMBINANT
3DNA-dependent RNA polymerase subunit rpo132RNA polymeraseCOMPLEX#21RECOMBINANT
4Nucleic acidsNucleic acidCOMPLEX#9-#111NATURAL
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus GLV-1h68502057
23Vaccinia virus GLV-1h68502057
34Vaccinia virus GLV-1h68502057
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus10245
23Vaccinia virus10245
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: SPOT SCAN
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: 1.02 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4958
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3CTF correction
5WarpCTF correction
8Cootmodel fitting
10PHENIXmodel refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 632458
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110733 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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