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- PDB-8ag3: Vaccinia C16 N-terminal domains -

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Basic information

Entry
Database: PDB / ID: 8ag3
TitleVaccinia C16 N-terminal domains
ComponentsProtein C10
KeywordsVIRAL PROTEIN / C16 vaccinia virus protein Ku70/Ku80 DNA binding inhibition
Function / homologyPoxvirus C4/C10 / Poxvirus C4/C10 protein / Protein C10
Function and homology information
Biological speciesVaccinia virus Western Reserve
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsRivera-Calzada, A. / Arribas-Bosacoma, R. / Pearl, L.H. / Llorca, O.
Funding support Spain, United Kingdom, 6items
OrganizationGrant numberCountry
Agencia Estatal de Investigacion (AEI)AEI/10.13039/501100011033 Spain
Ministerio de Ciencia e Innovacion (MCIN)PID2020-114429RB-I00 Spain
Autonomous Community of MadridY2018/BIO-4747 Spain
Autonomous Community of MadridP2018/NMT-4443 Spain
Cancer Research UKC302/A14532 United Kingdom
Cancer Research UKC302/A24386 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus.
Authors: Angel Rivera-Calzada / Raquel Arribas-Bosacoma / Alba Ruiz-Ramos / Paloma Escudero-Bravo / Jasminka Boskovic / Rafael Fernandez-Leiro / Antony W Oliver / Laurence H Pearl / Oscar Llorca /
Abstract: Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as ...Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.
History
DepositionJul 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Protein C10
D: Protein C10


Theoretical massNumber of molelcules
Total (without water)84,9532
Polymers84,9532
Non-polymers00
Water0
1


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

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Components

#1: Protein Protein C10


Mass: 42476.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus Western Reserve / Strain: Western Reserve / Gene: VACWR010, C10L, VACWR209 / Plasmid: pET-52b(+)
Details (production host): Original plasmid modified so MCS contains TEV-TwinStrep
Production host: Escherichia coli (E. coli) / Strain (production host): Rossetta2(DE3) / References: UniProt: P03296

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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: C16 N-terminal domains in the C16-Ku70/Ku80 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.230 MDa / Experimental value: NO
Source (natural)Organism: Vaccinia virus / Strain: Western Reserve
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rossetta2(DE3) / Plasmid: Modified pET-52b(+)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES-KOHHEPES-KOH1
2250 mMNaClSodium chlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
40.5 mMTCEPTCEP1
50.5 mMPMSFPMSF1
SpecimenConc.: 0.4 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 R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 278 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / 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) / Num. of real images: 13216
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1RELIONparticle selection
2Topazparticle selection
3EPUimage acquisition
5CTFFIND4.1CTF correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
13RELIONclassification
14cryoSPARCclassification
15RELION3D reconstruction
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 12495814
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81353 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial model for chains C and D was generated using AlphaFold2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0022676
ELECTRON MICROSCOPYf_angle_d0.5613610
ELECTRON MICROSCOPYf_dihedral_angle_d4.263350
ELECTRON MICROSCOPYf_chiral_restr0.043412
ELECTRON MICROSCOPYf_plane_restr0.007458

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