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- PDB-6rie: Structure of Vaccinia Virus DNA-dependent RNA polymerase co-trans... -

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Entry
Database: PDB / ID: 6rie
TitleStructure of Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional capping complex
Components
  • (DNA-dependent RNA polymerase subunit ...) x 4
  • (DNA-directed RNA polymerase ...) x 4
  • Large subunit of mRNA capping enzyme
  • Non-template DNA strand
  • RNA
  • Small subunit of mRNA capping enzyme
  • Template strand DNA
KeywordsVIRAL PROTEIN / Vaccinia / RNA polymerase / Transcription / Gene expression
Function / homology
Function and homology information


polynucleotide 5'-phosphatase / virion component => GO:0044423 / DNA-templated viral transcription / inorganic triphosphate phosphatase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide 5'-phosphatase activity / viral transcription / RNA polymerase I activity / DNA-directed RNA polymerase complex ...polynucleotide 5'-phosphatase / virion component => GO:0044423 / DNA-templated viral transcription / inorganic triphosphate phosphatase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide 5'-phosphatase activity / viral transcription / RNA polymerase I activity / DNA-directed RNA polymerase complex / DNA-templated transcription termination / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / mRNA guanylyltransferase activity / mRNA guanylyltransferase / mRNA (guanine-N7)-methyltransferase / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / DNA-templated transcription / GTP binding / DNA binding / RNA binding / zinc ion binding / metal ion binding
Similarity search - Function
Poxvirus mRNA capping enzyme, small subunit / mRNA-capping enzyme catalytic subunit, OB fold domain superfamily / mRNA-capping enzyme catalytic subunit, nucleotidyltransferase domain superfamily / mRNA-capping enzyme catalytic subunit, RNA triphosphatase domain superfamily / : / : / mRNA capping enzyme catalytic subunit, GTase, NTPase domain / mRNA-capping enzyme catalytic subunit, GTase, OB fold / Poxvirus mRNA capping enzyme, small subunit / Poxvirus mRNA capping enzyme, small subunit superfamily ...Poxvirus mRNA capping enzyme, small subunit / mRNA-capping enzyme catalytic subunit, OB fold domain superfamily / mRNA-capping enzyme catalytic subunit, nucleotidyltransferase domain superfamily / mRNA-capping enzyme catalytic subunit, RNA triphosphatase domain superfamily / : / : / mRNA capping enzyme catalytic subunit, GTase, NTPase domain / mRNA-capping enzyme catalytic subunit, GTase, OB fold / Poxvirus mRNA capping enzyme, small subunit / Poxvirus mRNA capping enzyme, small subunit superfamily / Poxvirus mRNA capping enzyme, small subunit / mRNA capping enzyme, large subunit, ATPase/guanylyltransferase, virus / mRNA capping enzyme catalytic subunit, RNA triphosphatase domain / 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 / mRNA cap guanine-N7 methyltransferase / mRNA (guanine-N(7))-methyltransferase domain / mRNA (guanine-N(7))-methyltransferase domain / mRNA (guanine-N(7)-)-methyltransferase (EC 2.1.1.56) domain profile. / 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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / 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 / GTP--RNA guanylyltransferase / DNA-directed RNA polymerase 18 kDa subunit ...S-ADENOSYLMETHIONINE / 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 / GTP--RNA guanylyltransferase / DNA-directed RNA polymerase 18 kDa subunit / Virus termination factor small subunit / DNA-directed RNA polymerase 19 kDa subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 147 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / mRNA-capping enzyme catalytic subunit / mRNA-capping enzyme regulatory subunit OPG124 / DNA-directed RNA polymerase 7 kDa subunit / DNA-directed RNA polymerase 22 kDa subunit / DNA-directed RNA polymerase 147 kDa polypeptide / DNA-directed RNA polymerase 133 kDa polypeptide / DNA-directed RNA polymerase 19 kDa subunit / DNA-directed RNA polymerase 18 kDa subunit
Similarity search - Component
Biological speciesVaccinia virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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
L: Small subunit of mRNA capping enzyme
N: Non-template DNA strand
O: Large subunit of mRNA capping enzyme
P: RNA
S: DNA-directed RNA polymerase 30 kDa polypeptide
T: Template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)581,76920
Polymers581,06113
Non-polymers7097
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area70440 Å2
ΔGint-370 kcal/mol
Surface area174020 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 147 / VACV094


Mass: 146995.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus
Gene: VAC_DPP10_109, VAC_DPP11_109, VAC_DPP13_109, VAC_DPP15_109, VAC_DPP19_109, VAC_DPP21_109, VACAC2_109, VACCL3_109, VACV_094, VACV_IOC_B141_124, VACV_IOC_B388_124, VACV_TT8_118
Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: Q1PIV1, UniProt: O57204*PLUS
#2: Protein DNA-dependent RNA polymerase subunit rpo132


Mass: 133526.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL194 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1Q1, UniProt: Q76ZP7*PLUS, DNA-directed RNA polymerase
#6: Protein DNA-dependent RNA polymerase subunit rpo18


Mass: 17917.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL147 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1K4, UniProt: Q76ZS0*PLUS, DNA-directed RNA polymerase
#7: Protein DNA-dependent RNA polymerase subunit rpo7


Mass: 7299.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL107 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1G3, UniProt: P68314*PLUS, 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 / Gene: GL205 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1R2, UniProt: O57233*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit


Mass: 21365.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL123 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1I0, UniProt: P68609*PLUS, 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 / Gene: GL167 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1M4, UniProt: Q76ZQ8*PLUS, DNA-directed RNA polymerase
#12: 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 / Gene: GL076 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1D1, UniProt: O57187*PLUS, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules LO

#8: Protein Small subunit of mRNA capping enzyme


Mass: 33396.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL155 / Production host: Vaccinia virus / Variant (production host): 1h439
References: UniProt: B9U1L2, UniProt: P04318*PLUS, mRNA (guanine-N7)-methyltransferase
#10: Protein Large subunit of mRNA capping enzyme


Mass: 96888.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: GL135 / Production host: Vaccinia virus / Variant (production host): 1h439 / References: UniProt: B9U1J2, UniProt: P04298*PLUS

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DNA chain , 2 types, 2 molecules NT

#9: DNA chain Non-template DNA strand


Mass: 14775.473 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus
#13: 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: 9822.829 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Production host: Enterobacteria phage T7 (virus)

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Non-polymers , 3 types, 7 molecules

#14: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#15: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#16: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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 co-transcriptional capping complexCOMPLEX#1-#130MULTIPLE SOURCES
2DNA-dependent RNA polymerase subunitCOMPLEX#1-#8, #10, #121RECOMBINANT
3RNACOMPLEX#111RECOMBINANT
4syntheticCOMPLEX#9, #131RECOMBINANT
Molecular weightValue: 0.58 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus10245
23Vaccinia virus10245
34Vaccinia virus10245
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Vaccinia virus10245
33Enterobacteria phage T7 (virus)10760
44synthetic construct (others)32630
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 FIELD / 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
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 632458
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77706 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 4CKB

4ckb

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