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- PDB-8qam: vaccinia virus Uracil DNA glycosidase mutant I197K-V200E-L204K -

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Basic information

Entry
Database: PDB / ID: 8qam
Titlevaccinia virus Uracil DNA glycosidase mutant I197K-V200E-L204K
ComponentsUracil-DNA glycosylase
KeywordsHYDROLASE / DNA POLYMERASE PROCESSIVITY FACTOR / DNA BINDING / DNA POLYMERASE BINDING / HYDROLASE-REPLICATION COMPLEX
Function / homologyuracil-DNA glycosylase / Uracil-DNA glycosylase, active site / Uracil-DNA glycosylase signature. / Uracil-DNA glycosylase-like domain superfamily / viral DNA genome replication / uracil DNA N-glycosylase activity / DNA repair / DNA binding / Uracil-DNA glycosylase
Function and homology information
Biological speciesVaccinia virus Copenhagen
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.32 Å
AuthorsTarbouriech, N. / Burmeister, W.P.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-13-BSV8-0014 France
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 23, 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
B: Uracil-DNA glycosylase
C: Uracil-DNA glycosylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,08912
Polymers51,1532
Non-polymers93710
Water13,295738
1
B: Uracil-DNA glycosylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,0456
Polymers25,5761
Non-polymers4685
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Uracil-DNA glycosylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,0456
Polymers25,5761
Non-polymers4685
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)161.668, 161.668, 39.281
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number79
Space group name H-MI4

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Components

#1: Protein Uracil-DNA glycosylase /


Mass: 25576.271 Da / Num. of mol.: 2 / Mutation: I197K-V200E-L204K
Source method: isolated from a genetically manipulated source
Details: first residues GAMGS are cloning atrifacts / Source: (gene. exp.) Vaccinia virus Copenhagen / Gene: OPG116 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 star / References: UniProt: P20536
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 738 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.51 Å3/Da / Density % sol: 50.97 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 22 % PEG4000, 100 mM LiSO4, 100 mM Hepes pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.97856 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 25, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97856 Å / Relative weight: 1
ReflectionResolution: 1.318→40.42 Å / Num. obs: 119652 / % possible obs: 99.5 % / Redundancy: 13.29 % / CC1/2: 0.999 / Rmerge(I) obs: 0.05 / Net I/σ(I): 23.4
Reflection shellResolution: 1.318→1.34 Å / Rmerge(I) obs: 1.134 / Num. unique obs: 5311 / CC1/2: 0.844 / % possible all: 90.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
Aimlessdata scaling
XDSdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.32→38.2 Å / Cor.coef. Fo:Fc: 0.982 / Cor.coef. Fo:Fc free: 0.974 / SU B: 1.52 / SU ML: 0.028 / Cross valid method: THROUGHOUT / ESU R: 0.044 / ESU R Free: 0.045 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.16463 6094 5.1 %RANDOM
Rwork0.12951 ---
obs0.13129 113557 99.46 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 22.741 Å2
Baniso -1Baniso -2Baniso -3
1-0.04 Å2-0 Å2-0 Å2
2--0.04 Å2-0 Å2
3----0.08 Å2
Refinement stepCycle: 1 / Resolution: 1.32→38.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3554 0 56 738 4348
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0134043
X-RAY DIFFRACTIONr_bond_other_d0.0020.0173767
X-RAY DIFFRACTIONr_angle_refined_deg2.1181.6485541
X-RAY DIFFRACTIONr_angle_other_deg1.6131.5818853
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.8685520
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.85923.054203
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.77515741
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.7561519
X-RAY DIFFRACTIONr_chiral_restr0.1370.2531
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.024506
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02835
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.5861.8561877
X-RAY DIFFRACTIONr_mcbond_other3.5871.8561876
X-RAY DIFFRACTIONr_mcangle_it4.282.7922380
X-RAY DIFFRACTIONr_mcangle_other4.2792.7922381
X-RAY DIFFRACTIONr_scbond_it5.7792.3982166
X-RAY DIFFRACTIONr_scbond_other5.7682.3932151
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.4673.3653105
X-RAY DIFFRACTIONr_long_range_B_refined7.25625.6894940
X-RAY DIFFRACTIONr_long_range_B_other6.90324.2714745
X-RAY DIFFRACTIONr_rigid_bond_restr5.26437809
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.32→1.352 Å
RfactorNum. reflection% reflection
Rfree0.476 454 -
Rwork0.467 7804 -
obs--93.38 %

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