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- PDB-8j86: Monkeypox virus DNA replication holoenzyme F8, A22 and E4 complex... -

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Basic information

Entry
Database: PDB / ID: 8j86
TitleMonkeypox virus DNA replication holoenzyme F8, A22 and E4 complex in a DNA binding form
Components
  • DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')
  • DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*TP*)-3')
  • DNA polymerase processivity factor component A20
  • DNA polymerase
  • E4R
KeywordsVIRAL PROTEIN / Monkeypox virus / DNA replication holoenzyme / DNA replication machinery / DNA polymerase / B-family DNA polymerase / uracil-DNA glycosylase / MPXV / orthopoxvirus / poxviridae / DNA processivity factor / DNA
Function / homology
Function and homology information


viral DNA genome replication / uracil DNA N-glycosylase activity / DNA recombination / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA repair / 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 polymerase family B signature. ...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 polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional 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: 3.22 Å
AuthorsXu, Y. / Wu, Y. / Wu, X. / Zhang, Y. / Yang, Y. / Li, D. / Yang, B. / Gao, K. / Zhang, Z. / Dong, C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32250710142 China
CitationJournal: To Be Published
Title: Structure of DNA replication machinery from human monkeypox virus in complex with a DNA duplex
Authors: Xu, Y.X. / Wu, Y.Q. / Wu, X.Y. / Zhang, Y.Y. / Yang, Y.X. / Li, D.Y. / Yang, B. / Gao, K. / Zhang, Z.Y. / Dong, C.J.
History
DepositionApr 30, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 1, 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 polymerase
B: E4R
C: DNA polymerase processivity factor component A20
P: DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')
T: DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*TP*)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)216,2938
Polymers216,1735
Non-polymers1203
Water0
1


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

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein DNA polymerase / / Mpox DNA polymerase F8


Mass: 120041.156 Da / Num. of mol.: 1 / Mutation: L108F, W411L
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 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
#3: 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

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DNA chain , 2 types, 2 molecules PT

#4: DNA chain DNA (5'-D(P*AP*GP*CP*TP*GP*CP*TP*AP*TP*GP*TP*GP*AP*GP*AP*TP*TP*AP*AP*GP*TP*TP*AP*T)-3')


Mass: 7438.825 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#5: DNA chain DNA (5'-D(P*GP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*GP*AP*TP*AP*AP*CP*TP*TP*AP*AP*TP*CP*TP*CP*AP*CP*AP*TP*AP*GP*CP*AP*GP*CP*TP*)-3')


Mass: 11605.480 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Non-polymers , 1 types, 3 molecules

#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca / 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

ComponentName: Monkeypox virus replication holoenzyme F8-A22-E4 complex with DNA duplex only
Type: COMPLEX / Details: F8-A22-E4 complex with DNA duplex only / Entity ID: #1-#5 / 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: 25 mM MOPS, pH 7.0, 200 mM NaCl, 5% (w/v) glycerol, 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 duplex only
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 FIELDBright-field microscopy / 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
12cryoSPARCclassification
13cryoSPARC3D reconstruction
Image processingDetails: The selected movies are corrected with motion correct
CTF correctionType: NONE
Particle selectionDetails: 15,300,849
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 960549 / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214539
ELECTRON MICROSCOPYf_angle_d0.57319800
ELECTRON MICROSCOPYf_dihedral_angle_d14.8582171
ELECTRON MICROSCOPYf_chiral_restr0.0592193
ELECTRON MICROSCOPYf_plane_restr0.0032397

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