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- PDB-8p0j: Cryo EM map and model of the vaccinia RNA polymerase intermediate... -

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Basic information

Entry
Database: PDB / ID: 8p0j
TitleCryo EM map and model of the vaccinia RNA polymerase intermediate pre-initiation open promoter complex
Components
  • (DNA-directed RNA polymerase ...) x 8
  • (Intermediate transcription factor 3 ...) x 2
  • (mRNA-capping enzyme ...) x 2
  • Non-template DNA oligomer
  • Template DNA oligomer
KeywordsTRANSCRIPTION / RNA polymerase / RNAP / Vaccinia / MPox / PIC / pre-initiation complex
Function / homology
Function and homology information


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 ...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
VITF-3 subunit protein / Poxvirus intermediate transcription factor / VITF-3 subunit protein / Poxvirus intermediate transcription factor / 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 ...VITF-3 subunit protein / Poxvirus intermediate transcription factor / VITF-3 subunit protein / Poxvirus intermediate transcription factor / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase 147 kDa polypeptide / mRNA-capping enzyme catalytic subunit / mRNA-capping enzyme regulatory subunit OPG124 / Intermediate transcription factor 3 small subunit / Intermediate transcription factor 3 large subunit / DNA-directed RNA polymerase 18 kDa subunit / DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit ...DNA / DNA (> 10) / DNA-directed RNA polymerase 147 kDa polypeptide / mRNA-capping enzyme catalytic subunit / mRNA-capping enzyme regulatory subunit OPG124 / Intermediate transcription factor 3 small subunit / Intermediate transcription factor 3 large subunit / DNA-directed RNA polymerase 18 kDa subunit / DNA-directed RNA polymerase 30 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase 7 kDa subunit / 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsGrimm, C. / Jungwirth, S. / Fischer, U.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: To Be Published
Title: Cryo EM map and model of the vaccinia RNA polymerase intermediate pre-initiation open promoter complex (CASP target)
Authors: Grimm, C. / Jungwirth, S. / Fischer, U.
History
DepositionMay 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase 147 kDa polypeptide
B: DNA-directed RNA polymerase 133 kDa polypeptide
C: DNA-directed RNA polymerase 35 kDa subunit
E: DNA-directed RNA polymerase 22 kDa subunit
F: DNA-directed RNA polymerase 19 kDa subunit
G: DNA-directed RNA polymerase 18 kDa subunit
H: Intermediate transcription factor 3 large subunit
J: DNA-directed RNA polymerase 7 kDa subunit
L: mRNA-capping enzyme regulatory subunit OPG124
N: Non-template DNA oligomer
O: mRNA-capping enzyme catalytic subunit
S: DNA-directed RNA polymerase 30 kDa polypeptide
T: Template DNA oligomer
I: Intermediate transcription factor 3 small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)663,50719
Polymers663,22114
Non-polymers2865
Water9,152508
1


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

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Components

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

#1: Protein DNA-directed RNA polymerase 147 kDa polypeptide


Mass: 146995.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG105, RPO147, J6R / Production host: Vaccinia virus / References: UniProt: P20504, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase 133 kDa polypeptide


Mass: 133526.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG151, RPO132, A24R / Production host: Vaccinia virus / References: UniProt: P68694, DNA-directed RNA polymerase
#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: OPG156, RPO35, A29L / Production host: Vaccinia virus / References: UniProt: P21087, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase 22 kDa subunit


Mass: 21365.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MNQYNVKYLAKILCLKTEIARDPYAVINRNVLLRYTTDIEYNDLVTLITVRHKIDSMKTVFQVFNESSIN YTPVDDDYGEPIIITSYLQKGHNKFPVNFLYIDVVISDLFPSFVRLDTTETNIVNSVLQTGDGKKTLRLP KMLETEIVVKILYRPNIPLKIVRFFRNNMVTGVEIADRSVISVAD
Source: (gene. exp.) Vaccinia virus / Gene: OPG103, RPO22, J4R / Production host: Vaccinia virus / References: UniProt: P68608, 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: OPG131, RPO19, A5R / Production host: Vaccinia virus / References: UniProt: P68610, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase 18 kDa subunit


Mass: 17917.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG119, RPO18, D7R / Production host: Vaccinia virus / References: UniProt: P21034, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase 7 kDa subunit


Mass: 7299.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG090, RPO7, G5.5R / Production host: Vaccinia virus / References: UniProt: P68315, 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: OPG066, RPO30, E4L / Production host: Vaccinia virus / References: UniProt: P21082, DNA-directed RNA polymerase

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Intermediate transcription factor 3 ... , 2 types, 2 molecules HI

#7: Protein Intermediate transcription factor 3 large subunit / VITF-3 45 kDa subunit


Mass: 44662.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG150, VITF3L, A23R / Production host: Vaccinia virus / References: UniProt: P20998
#14: Protein Intermediate transcription factor 3 small subunit / VITF-3 32 kDa subunit / VITF-3 small subunit


Mass: 33618.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG134, VITF3S, A8R / Production host: Vaccinia virus / References: UniProt: P20986

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MRNA-capping enzyme ... , 2 types, 2 molecules LO

#9: Protein mRNA-capping enzyme regulatory subunit OPG124 / Virus termination factor small subunit / VTF small subunit / mRNA-capping enzyme 33 kDa subunit / ...Virus termination factor small subunit / VTF small subunit / mRNA-capping enzyme 33 kDa subunit / mRNA-capping enzyme D12 subunit / mRNA-capping enzyme small subunit


Mass: 33396.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG124, D12L / Production host: Vaccinia virus / References: UniProt: P20980
#11: Protein mRNA-capping enzyme catalytic subunit / Virus termination factor large subunit / VTF large subunit / mRNA-capping enzyme 97 kDa subunit / ...Virus termination factor large subunit / VTF large subunit / mRNA-capping enzyme 97 kDa subunit / mRNA-capping enzyme D1 subunit / mRNA-capping enzyme large subunit


Mass: 96888.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Gene: OPG113, D1R / Production host: Vaccinia virus
References: UniProt: P20979, mRNA 5'-phosphatase, mRNA guanylyltransferase, mRNA (guanine-N7)-methyltransferase

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DNA chain , 2 types, 2 molecules NT

#10: DNA chain Non-template DNA oligomer


Mass: 21587.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Production host: synthetic construct (others)
#13: DNA chain Template DNA oligomer


Mass: 21675.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus / Production host: synthetic construct (others)

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Non-polymers , 3 types, 513 molecules

#15: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#17: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 508 / Source method: isolated from a natural source / Formula: H2O

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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
1Vaccinia DNA-directed RNA polymerase open promoter intermediate pre-initiation complexCOMPLEX#1-#140RECOMBINANT
2Vaccinia DNA-directed RNA polymerase open promoter intermediate pre-initiation complexCOMPLEX#1-#9, #11-#12, #141RECOMBINANT
3Non-template DNA oligomer and Template DNA oligomerCOMPLEX#10, #131RECOMBINANT
Molecular weightValue: 0.75 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Vaccinia virus10245
33Vaccinia virus10245
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus10245
23Synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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 / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_4746refinement
PHENIXdev_4746refinement
EM software
IDNameVersionCategory
1cryoSPARC4.1particle selection
4cryoSPARC4.1CTF correction
10cryoSPARC4.1initial Euler assignment
13cryoSPARC4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9318600
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1421158 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 55.95 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00244163
ELECTRON MICROSCOPYf_angle_d0.41359984
ELECTRON MICROSCOPYf_chiral_restr0.04086815
ELECTRON MICROSCOPYf_plane_restr0.00277330
ELECTRON MICROSCOPYf_dihedral_angle_d11.097416728

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