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- PDB-8c8h: Cryo EM structure of the vaccinia complete RNA polymerase complex... -

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Basic information

Entry
Database: PDB / ID: 8c8h
TitleCryo EM structure of the vaccinia complete RNA polymerase complex lacking the capping enzyme
Components
  • (DNA-directed RNA polymerase ...Polymerase) x 8
  • Core protein E11
  • Early transcription factor 82 kDa subunit
  • Nucleoside triphosphatase I
  • RNA polymerase-associated transcription-specificity factor RAP94
  • chr17.trna16-GlnTTG
KeywordsVIRAL PROTEIN / RNA polymerase / RNAP / DNA-dependent RNA polymerase
Function / homology
Function and homology information


DNA-templated viral transcription / ATP-dependent chromatin remodeler activity / viral transcription / ribonucleoside triphosphate phosphatase activity / DNA-directed RNA polymerase complex / virion component / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...DNA-templated viral transcription / ATP-dependent chromatin remodeler activity / viral transcription / ribonucleoside triphosphate phosphatase activity / DNA-directed RNA polymerase complex / virion component / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / nucleoside-triphosphate phosphatase / host cell cytoplasm / DNA-binding transcription factor activity / DNA-templated transcription / positive regulation of DNA-templated transcription / DNA binding / zinc ion binding / ATP binding / metal ion binding
Similarity search - Function
Virion core protein, vaccinia E11L type / Chordopoxvirus E11 protein / Poxvirus early transcription factor, large subunit / Nucleoside triphosphatase I, C-terminal / Poxvirus early transcription factor (VETF), large subunit / Nucleoside triphosphatase I C-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 ...Virion core protein, vaccinia E11L type / Chordopoxvirus E11 protein / Poxvirus early transcription factor, large subunit / Nucleoside triphosphatase I, C-terminal / Poxvirus early transcription factor (VETF), large subunit / Nucleoside triphosphatase I C-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 / SNF2, N-terminal / SNF2-related domain / 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 / Helicase conserved C-terminal domain / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / 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 / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / DNA-directed RNA polymerase 18 kDa subunit / Nucleoside triphosphatase I / DNA-directed RNA polymerase 147 kDa polypeptide / Early transcription factor 82 kDa subunit / DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase 7 kDa subunit / RNA polymerase-associated transcription-specificity factor RAP94 ...RNA / RNA (> 10) / DNA-directed RNA polymerase 18 kDa subunit / Nucleoside triphosphatase I / DNA-directed RNA polymerase 147 kDa polypeptide / Early transcription factor 82 kDa subunit / DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase 7 kDa subunit / RNA polymerase-associated transcription-specificity factor RAP94 / Core protein OPG073 / DNA-directed RNA polymerase 22 kDa subunit / DNA-directed RNA polymerase 19 kDa subunit / DNA-directed RNA polymerase 133 kDa polypeptide
Similarity search - Component
Biological speciesVaccinia virus GLV-1h68
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsGrimm, G. / Bartuli, J. / Fischer, U.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)Fi573/23-1 Germany
Volkswagen Foundation9B813 Germany
Citation
Journal: To Be Published
Title: Cryo EM structure of the vaccinia complete RNA polymerase complex lacking the capping enzyme
Authors: Grimm, G. / Bartuli, J. / Fischer, U.
#1: Journal: Cell / Year: 2019
Title: Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.
Authors: Clemens Grimm / Hauke S Hillen / Kristina Bedenk / Julia Bartuli / Simon Neyer / Qian Zhang / Alexander Hüttenhofer / Matthias Erlacher / Christian Dienemann / Andreas Schlosser / Henning ...Authors: Clemens Grimm / Hauke S Hillen / Kristina Bedenk / Julia Bartuli / Simon Neyer / Qian Zhang / Alexander Hüttenhofer / Matthias Erlacher / Christian Dienemann / Andreas Schlosser / Henning Urlaub / Bettina Böttcher / Aladar A Szalay / Patrick Cramer / Utz Fischer /
Abstract: Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes ...Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNA. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.
History
DepositionJan 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: DNA-directed RNA polymerase 133 kDa polypeptide
E: DNA-directed RNA polymerase 22 kDa subunit
F: DNA-directed RNA polymerase 19 kDa subunit
G: DNA-directed RNA polymerase 18 kDa subunit
I: RNA polymerase-associated transcription-specificity factor RAP94
J: DNA-directed RNA polymerase 7 kDa subunit
R: Core protein E11
K: Early transcription factor 82 kDa subunit
U: chr17.trna16-GlnTTG
A: DNA-directed RNA polymerase 147 kDa polypeptide
Q: Core protein E11
Y: Nucleoside triphosphatase I
C: DNA-directed RNA polymerase 35 kDa subunit
S: DNA-directed RNA polymerase 30 kDa polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)713,38519
Polymers713,09914
Non-polymers2865
Water1,09961
1


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

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Components

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DNA-directed RNA polymerase ... , 8 types, 8 molecules BEFGJACS

#1: Protein DNA-directed RNA polymerase 133 kDa polypeptide


Mass: 133526.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q76ZP7, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase 22 kDa subunit


Mass: 21365.740 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P68609, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase 19 kDa subunit


Mass: 19020.088 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P68611, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase 18 kDa subunit


Mass: 17917.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P04310, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase 7 kDa subunit


Mass: 7299.715 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P68317, DNA-directed RNA polymerase
#10: Protein DNA-directed RNA polymerase 147 kDa polypeptide


Mass: 146995.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P07392, DNA-directed RNA polymerase
#12: Protein DNA-directed RNA polymerase 35 kDa subunit


Mass: 35430.676 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P24757, DNA-directed RNA polymerase
#13: Protein DNA-directed RNA polymerase 30 kDa polypeptide


Mass: 30074.293 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P21603, DNA-directed RNA polymerase

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Protein , 4 types, 5 molecules IRQKY

#5: Protein RNA polymerase-associated transcription-specificity factor RAP94 / Protein H4 / RPO-associated protein of 94 kDa


Mass: 93667.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P68438
#7: Protein Core protein E11


Mass: 14914.090 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P68448
#8: Protein Early transcription factor 82 kDa subunit / ETF large subunit / VETF A7 subunit / Vaccinia virus early transcription factor large subunit / ...ETF large subunit / VETF A7 subunit / Vaccinia virus early transcription factor large subunit / VETF large subunit


Mass: 82398.906 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: P20636
#11: Protein Nucleoside triphosphatase I / Factor X / NPH-I / Nucleoside triphosphate phosphohydrolase I / NPH I


Mass: 72465.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68
References: UniProt: P05807, nucleoside-triphosphate phosphatase

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RNA chain , 1 types, 1 molecules U

#9: RNA chain chr17.trna16-GlnTTG


Mass: 23108.650 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68

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Non-polymers , 3 types, 66 molecules

#14: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#15: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#16: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 61 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: Vaccinia complete RNA polymerase complex lacking the capping enzyme
Type: COMPLEX / Entity ID: #1-#13 / Source: NATURAL
Molecular weightValue: 0.7 MDa / Experimental value: YES
Source (natural)Organism: Vaccinia virus GLV-1h68
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_4746refinement
PHENIXdev_4746refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21338 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 148.17 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002342203
ELECTRON MICROSCOPYf_angle_d0.552757346
ELECTRON MICROSCOPYf_chiral_restr0.04246552
ELECTRON MICROSCOPYf_plane_restr0.00597012
ELECTRON MICROSCOPYf_dihedral_angle_d14.024616033

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