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- PDB-8hpa: Monkeypox virus DNA replication holoenzyme F8, A22 and E4 complex... -

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Basic information

Entry
Database: PDB / ID: 8hpa
TitleMonkeypox virus DNA replication holoenzyme F8, A22 and E4 complex in a DNA binding form
Components
  • DNA (5'-D(*CP*GP*AP*TP*CP*CP*TP*TP*CP*CP*CP*CP*TP*AP*C)-3')
  • DNA (5'-D(P*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*AP*GP*GP*AP*TP*CP*G)-3')
  • DNA polymerase
  • DNA polymerase processivity factor component A20
  • E4R
KeywordsVIRAL PROTEIN / Monkeypox virus / DNA replication holoenzyme / DNA replication machinery / F8 / A22 / E4 / DNA polymerase / B-family DNA polymerase / uracil-DNA glycosylase / MPXV / orthopoxvirus / poxviridae / F8-A22-E4 complex / DNA processivity factor
Function / homology
Function and homology information


uracil DNA N-glycosylase activity / viral DNA genome replication / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / DNA repair / nucleotide binding / DNA binding
Similarity search - Function
DNA-directed DNA polymerase, family B, viral insert domain / DNA polymerase B exonuclease, N-terminal / DNA polymerase family B viral insert / DNA polymerase family B exonuclease domain, N-terminal / Chordopoxvirus A20R / Chordopoxvirus A20R protein / Uracil-DNA glycosylase, active site / Uracil-DNA glycosylase signature. / Uracil-DNA glycosylase-like domain superfamily / : ...DNA-directed DNA polymerase, family B, viral insert domain / DNA polymerase B exonuclease, N-terminal / DNA polymerase family B viral insert / DNA polymerase family B exonuclease domain, N-terminal / Chordopoxvirus A20R / Chordopoxvirus A20R protein / Uracil-DNA glycosylase, active site / Uracil-DNA glycosylase signature. / Uracil-DNA glycosylase-like domain superfamily / : / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA polymerase processivity factor / Uracil-DNA glycosylase / DNA polymerase
Similarity search - Component
Biological speciesMonkeypox virus
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsXu, Y. / Wu, Y. / Zhang, Y. / Fan, R. / Yang, Y. / Li, D. / Yang, B. / Zhang, Z. / Dong, C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32250710142 China
CitationJournal: Cell Res / Year: 2023
Title: Cryo-EM structures of human monkeypox viral replication complexes with and without DNA duplex.
Authors: Yunxia Xu / Yaqi Wu / Yuanyuan Zhang / Ruixin Fan / Yaxue Yang / Danyang Li / Shimin Zhu / Biao Yang / Zhengyu Zhang / Changjiang Dong /
History
DepositionDec 12, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.0Jan 31, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Sep 18, 2024Group: Data collection / Database references
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase
C: DNA polymerase processivity factor component A20
B: E4R
E: DNA (5'-D(P*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*AP*GP*GP*AP*TP*CP*G)-3')
D: DNA (5'-D(*CP*GP*AP*TP*CP*CP*TP*TP*CP*CP*CP*CP*TP*AP*C)-3')


Theoretical massNumber of molelcules
Total (without water)212,1995
Polymers212,1995
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein DNA polymerase


Mass: 120041.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus / Gene: F8L
Production host: Insect expression vector pBlueBachsGCA1 (others)
References: UniProt: Q5IXW8
#2: Protein DNA polymerase processivity factor component A20


Mass: 49203.926 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus / Gene: A22R
Production host: Insect expression vector pBlueBachsGCA1 (others)
References: UniProt: Q5IXP2
#3: Protein E4R


Mass: 27883.709 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus / Gene: E4R
Production host: Insect expression vector pBlueBachsGCA1 (others)
References: UniProt: Q5IXS4, uracil-DNA glycosylase
#4: DNA chain DNA (5'-D(P*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*AP*GP*GP*AP*TP*CP*G)-3')


Mass: 8148.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#5: DNA chain DNA (5'-D(*CP*GP*AP*TP*CP*CP*TP*TP*CP*CP*CP*CP*TP*AP*C)-3')


Mass: 6921.475 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
Has protein modificationN

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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: Monkeypox virus replication holoenzyme F8-A22-E4 complex with DNA
Type: COMPLEX / Details: F8-A22-E4 complex with DNA / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 191 kDa/nm / Experimental value: NO
Source (natural)Organism: Monkeypox virus
Source (recombinant)Organism: Baculovirus expression vector pFastBac1-HM
Buffer solutionpH: 8
Details: 25mM HEPES pH 8.0, 300mM NaCl, 5% (w/v) glycerol, and 1 mM TCEP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This is a complex of F8-A22-E4 and DNA
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU9.03image acquisition
4cryoSPARCCTF correction
7CCP4 package9.03model fitting
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
Image processingDetails: The selected movies are corrected with motion correct
CTF correctionType: NONE
Particle selectionDetails: 1063499
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72237 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314502
ELECTRON MICROSCOPYf_angle_d0.52919739
ELECTRON MICROSCOPYf_dihedral_angle_d13.9972145
ELECTRON MICROSCOPYf_chiral_restr0.0432185
ELECTRON MICROSCOPYf_plane_restr0.0042399

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