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- PDB-8k8s: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP -

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Basic information

Entry
Database: PDB / ID: 8k8s
TitleF8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
Components
  • (DNA polymerase ...) x 2
  • DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
  • DNA (5'-D(P*TP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')
  • Uracil-DNA glycosylase E4
KeywordsVIRAL PROTEIN/DNA / RECOMBINATION / REPLICATION / VIRAL PROTEIN-DNA COMPLEX
Function / homologyChordopoxvirus A20R / Chordopoxvirus A20R protein / DNA replication / Chem-HF4 / DNA / DNA (> 10) / DNA polymerase processivity factor
Function and homology information
Biological speciesMonkeypox virus
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsShen, Y.P. / Li, Y.N. / Yan, R.H.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0509301 China
CitationJournal: Structure / Year: 2024
Title: Structural basis for the inhibition mechanism of the DNA polymerase holoenzyme from mpox virus.
Authors: Yaping Shen / Yaning Li / Renhong Yan /
Abstract: There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is ...There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases.
History
DepositionJul 31, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase F8
B: Uracil-DNA glycosylase E4
C: DNA polymerase processivity factor component A20
E: DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
F: DNA (5'-D(P*TP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,4507
Polymers199,9425
Non-polymers5072
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA polymerase ... , 2 types, 2 molecules AC

#1: Protein DNA polymerase F8


Mass: 117045.055 Da / Num. of mol.: 1 / Mutation: D166A, E168A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#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 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q5IXP2

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Protein , 1 types, 1 molecules B

#2: Protein Uracil-DNA glycosylase E4


Mass: 25107.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus / Cell line (production host): HEK293 / Production host: Homo sapiens (human)

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DNA chain , 2 types, 2 molecules EF

#4: DNA chain DNA (5'-D(*AP*TP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')


Mass: 3518.307 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#5: DNA chain DNA (5'-D(P*TP*AP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*AP*T)-3')


Mass: 5067.301 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Non-polymers , 3 types, 3 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-HF4 / 4-amino-1-{5-O-[(S)-hydroxy{[(R)-hydroxy(phosphonooxy)phosphoryl]oxy}phosphoryl]-beta-D-arabinofuranosyl}pyrimidin-2(1H)-one / Cytarabine-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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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: F8-A22-E4 complex of MPXV in complex with DNA and Ara-CTP
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Monkeypox virus
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1400 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 4 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 326139 / Symmetry type: POINT

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