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- PDB-8hoy: Cryo-EM structure of monkeypox virus DNA replication holoenzyme F... -

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Basic information

Entry
Database: PDB / ID: 8hoy
TitleCryo-EM structure of monkeypox virus DNA replication holoenzyme F8, A22 and E4 complex without DNA at 2.76 angostram
Components
  • DNA polymerase
  • DNA polymerase processivity factor component A20
  • E4R
KeywordsVIRAL PROTEIN / DNA replication machinery / DNA polymerase / B-family DNA polymerase / uracil-DNA glycosylase / MPXV / orthopoxvirus / poxviridae / DNA processivity factor
Function / homology
Function and homology information


viral DNA genome replication / uracil DNA N-glycosylase activity / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / SOS response / base-excision repair, gap-filling / DNA recombination / DNA replication / DNA-directed DNA polymerase ...viral DNA genome replication / uracil DNA N-glycosylase activity / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / SOS response / base-excision repair, gap-filling / DNA recombination / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / 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 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 polymerase processivity factor / Uracil-DNA glycosylase / DNA polymerase
Similarity search - Component
Biological speciesMonkeypox virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 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: To Be Published
Title: Structure of DNA replication machinery from human monkeypox virus
Authors: Xu, Y.X. / Wu, Y.Q. / Zhang, Y.Y. / Fan, R.X. / Yang, Y.X. / Li, D.Y. / Yang, B. / Zhang, Z.Y. / Dong, C.J.
History
DepositionDec 11, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA polymerase
C: DNA polymerase processivity factor component A20
B: E4R


Theoretical massNumber of molelcules
Total (without water)197,1293
Polymers197,1293
Non-polymers00
Water00
1


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

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Components

#1: Protein DNA polymerase


Mass: 120041.156 Da / Num. of mol.: 1 / Mutation: L108F, W411L
Source method: isolated from a genetically manipulated source
Details: MPXV DNA replication machinery catalytic subunit F8
Source: (gene. exp.) Monkeypox virus / Gene: F8L
Production host: Insect expression vector pBlueBacmsGCA1His (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

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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 without DNA
Type: COMPLEX / Details: F8-A22-E4 complex / 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
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: 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

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU9.03image acquisition
4cryoSPARCCTF correction
12cryoSPARCclassification
13cryoSPARC3D reconstruction
Image processingDetails: The selected movies are corrected with motion correct
CTF correctionType: NONE
Particle selectionDetails: 11,662,000
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 343570 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213746
ELECTRON MICROSCOPYf_angle_d0.4418576
ELECTRON MICROSCOPYf_dihedral_angle_d3.6341822
ELECTRON MICROSCOPYf_chiral_restr0.0412054
ELECTRON MICROSCOPYf_plane_restr0.0042366

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