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- PDB-8q3r: Cryo-EM structure of the DNA polymerase holoenzyme E9-A20-D4 of v... -

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Basic information

Entry
Database: PDB / ID: 8q3r
TitleCryo-EM structure of the DNA polymerase holoenzyme E9-A20-D4 of vaccinia virus
Components
  • DNA polymerase
  • DNA polymerase processivity factor component OPG148
  • Uracil-DNA glycosylase
KeywordsVIRAL PROTEIN / DNA polymerase / holoenzyme / processivity factor / uracil-DNA glycosylase
Function / homology
Function and homology information


uracil-DNA glycosylase / uracil DNA N-glycosylase activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / hydrolase activity / DNA repair ...uracil-DNA glycosylase / uracil DNA N-glycosylase activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / hydrolase 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-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 polymerase / Uracil-DNA glycosylase / DNA polymerase processivity factor component OPG148
Similarity search - Component
Biological speciesVaccinia virus Copenhagen
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBurmeister, W.P. / Ballandras-Colas, A. / Boettcher, B. / Grimm, C.
Funding support France, United States, Germany, 7items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-22-CE11-0007-01 France
Agence Nationale de la Recherche (ANR)and ANR-13-BSV8-0014 France
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-0002 France
Agence Nationale de la Recherche (ANR)ANR-10-INBS-0005-02 France
Agence Nationale de la Recherche (ANR)ANR-17-EURE-0003 France
National Institutes of Health/National Center for Research Resources (NIH/NCRR)P41-GM103311 United States
German Research Foundation (DFG)Fi573/22-1 Germany
CitationJournal: PLoS Pathog / Year: 2024
Title: Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.
Authors: Wim P Burmeister / Laetitia Boutin / Aurelia C Balestra / Henri Gröger / Allison Ballandras-Colas / Stephanie Hutin / Christian Kraft / Clemens Grimm / Bettina Böttcher / Utz Fischer / ...Authors: Wim P Burmeister / Laetitia Boutin / Aurelia C Balestra / Henri Gröger / Allison Ballandras-Colas / Stephanie Hutin / Christian Kraft / Clemens Grimm / Bettina Böttcher / Utz Fischer / Nicolas Tarbouriech / Frédéric Iseni /
Abstract: The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the ...The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.
History
DepositionAug 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Uracil-DNA glycosylase
A: DNA polymerase processivity factor component OPG148
E: DNA polymerase


Theoretical massNumber of molelcules
Total (without water)197,0773
Polymers197,0773
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, SAXS, MW=177-221 kDa, light scattering, MW=185 kDa
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Uracil-DNA glycosylase


Mass: 27468.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: D4 carries a N-terminal Strep-tag / Source: (gene. exp.) Vaccinia virus Copenhagen / Gene: OPG116 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P20536
#2: Protein DNA polymerase processivity factor component OPG148


Mass: 49247.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus Copenhagen / Gene: OPG148, A20R / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P20995
#3: Protein DNA polymerase


Mass: 120361.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus Copenhagen / Gene: OPG071 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P20509

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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: E9-A20-D4 DNA polymerase holoenzyme / Type: COMPLEX / Details: heterotrimer / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 196.825 kDa/nm / Experimental value: NO
Source (natural)Organism: Vaccinia virus / Strain: Copenhagen
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: High five
Buffer solutionpH: 7.6
Details: 35 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 3.8 mM desthiobiotin
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
235 mMTris-HClC4H15Cl3NO31
35 mMEDTAC10H16N2O81
43.8 mMdesthiobiotinC10H18N2O31
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 42000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 68 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1376
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.2.1particle selectionBlob picker
2EPUimage acquisition
4cryoSPARCv4.2.1CTF correctionPatch CTF
7UCSF Chimera1.15model fitting
9cryoSPARCv4.2.1initial Euler assignment2D Class
10cryoSPARCv4.2.1final Euler assignmentAb initio
11cryoSPARCv4.2.1classificationHetero Refine
12cryoSPARCv4.2.13D reconstructionNU-Refine
19PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 381864
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104239 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: Phenix real-space refinement
Atomic model building

3D fitting-ID: 1 / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDDetailsInitial refinement model-IDSource name
14od8

4od8

D4od8Dalso A1-A461PDB
2PDBDEV_00000075Ealso A304-A4262Other
38hg1

8hg1

A8hg1Aonly A47-A3033PDB
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 32.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004912180
ELECTRON MICROSCOPYf_angle_d0.597416467
ELECTRON MICROSCOPYf_chiral_restr0.04271823
ELECTRON MICROSCOPYf_plane_restr0.00422097
ELECTRON MICROSCOPYf_dihedral_angle_d13.72634568

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