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- PDB-8wpk: Structure of monkeypox virus polymerase complex F8-A22-E4-H5 with... -

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Basic information

Entry
Database: PDB / ID: 8wpk
TitleStructure of monkeypox virus polymerase complex F8-A22-E4-H5 with exgenous DNA
Components
  • DNA polymerase processivity factor
  • DNA polymerase
  • E4R Uracil-DNA glycosylase, DNA polymerase processivity factor
  • H5R late gene transcription factor
  • Primer DNA
  • Template DNA
KeywordsVIRAL PROTEIN / Viral DNA replication / monkeypox virus / polymerase / H5
Function / homology2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesMonkeypox virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWang, X. / Li, N. / Gao, N.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Mol Cell / Year: 2023
Title: Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.
Authors: Xiaohan Wang / Liangwen Ma / Ningning Li / Ning Gao /
Abstract: The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. ...The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.
History
DepositionOct 10, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 27, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA polymerase
B: DNA polymerase processivity factor
C: E4R Uracil-DNA glycosylase, DNA polymerase processivity factor
D: H5R late gene transcription factor
E: H5R late gene transcription factor
F: H5R late gene transcription factor
G: H5R late gene transcription factor
H: Primer DNA
I: Template DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,17911
Polymers310,6899
Non-polymers4902
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 7 molecules ABCDEFG

#1: Protein DNA polymerase /


Mass: 117147.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GenBank: URK20494.1 / Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Protein DNA polymerase processivity factor


Mass: 50466.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GenBank: URK20570.1 / Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#3: Protein E4R Uracil-DNA glycosylase, DNA polymerase processivity factor


Mass: 25107.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GenBank: URK20538.1 / Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#4: Protein
H5R late gene transcription factor / Ca2+-binding motif H5R VLTF-4 / late transcription factor 4


Mass: 23107.074 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: GenBank: URK20532.1 / Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293F / Production host: Homo sapiens (human)

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DNA chain , 2 types, 2 molecules HI

#5: DNA chain Primer DNA


Mass: 10802.956 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain Template DNA


Mass: 14736.490 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-D3T / 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE


Type: DNA OH 3 prime terminus / Mass: 466.169 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O13P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / 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
1F8-A22-E4-H5 with exogenous DNACOMPLEX#1-#60MULTIPLE SOURCES
2F8-A22-E4-H5COMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Monkeypox virus10244
33Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 59.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1301000 / Symmetry type: POINT

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