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- PDB-8ag5: Vaccinia C16 protein bound to Ku70/Ku80 -

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Basic information

Entry
Database: PDB / ID: 8ag5
TitleVaccinia C16 protein bound to Ku70/Ku80
Components
  • Ku70-Xrcc6
  • Protein C10
  • X-ray repair cross-complementing protein 5
KeywordsVIRAL PROTEIN / C16 vaccinia virus protein Ku70/Ku80 DNA binding inhibition
Function / homology
Function and homology information


DNA-dependent protein kinase complex / Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / cellular response to X-ray / regulation of smooth muscle cell proliferation ...DNA-dependent protein kinase complex / Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / cellular response to X-ray / regulation of smooth muscle cell proliferation / double-strand break repair via classical nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / nuclear telomere cap complex / IRF3-mediated induction of type I IFN / U3 snoRNA binding / regulation of telomere maintenance / recombinational repair / cellular hyperosmotic salinity response / protein localization to chromosome, telomeric region / 2-LTR circle formation / telomeric repeat DNA binding / DNA 3'-5' helicase / 5'-deoxyribose-5-phosphate lyase activity / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / telomere maintenance via telomerase / activation of innate immune response / telomere maintenance / cyclin binding / DNA helicase activity / DNA-(apurinic or apyrimidinic site) lyase / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / site of DNA damage / small-subunit processome / Nonhomologous End-Joining (NHEJ) / cellular response to gamma radiation / protein-DNA complex / double-strand break repair via nonhomologous end joining / enzyme activator activity / double-strand break repair / transcription regulator complex / scaffold protein binding / double-stranded DNA binding / DNA recombination / secretory granule lumen / ficolin-1-rich granule lumen / damaged DNA binding / chromosome, telomeric region / transcription cis-regulatory region binding / ribonucleoprotein complex / innate immune response / negative regulation of DNA-templated transcription / ubiquitin protein ligase binding / Neutrophil degranulation / DNA damage response / nucleolus / positive regulation of DNA-templated transcription / protein-containing complex binding / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / protein-containing complex / DNA-templated transcription / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / cytosol
Similarity search - Function
Poxvirus C4/C10 / Poxvirus C4/C10 protein / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm ...Poxvirus C4/C10 / Poxvirus C4/C10 protein / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 N-terminal alpha/beta domain / Ku70/Ku80 beta-barrel domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / von Willebrand factor, type A domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Protein C10 / DNA repair protein Ku70 / DNA repair protein Ku80
Similarity search - Component
Biological speciesHomo sapiens (human)
Vaccinia 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: Data collection / Database references
Category: citation / citation_author / pdbx_initial_refinement_model
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
Revision 1.2Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ku70-Xrcc6
B: X-ray repair cross-complementing protein 5
C: Protein C10
D: Protein C10


Theoretical massNumber of molelcules
Total (without water)244,7354
Polymers244,7354
Non-polymers00
Water00
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 Ku70-Xrcc6


Mass: 74234.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pFASTBAC-HTb / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P12956
#2: Protein X-ray repair cross-complementing protein 5 / 86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase ...86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase II 80 kDa subunit / CTC box-binding factor 85 kDa subunit / CTC85 / CTCBF / DNA repair protein XRCC5 / Ku80 / Ku86 / Lupus Ku autoantigen protein p86 / Nuclear factor IV / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)


Mass: 85546.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Plasmid: pFASTBAC-HTb / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: 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: Vaccinia C16 protein bound to Ku70/Ku80 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.230 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES-KOHHEPES-KOH1
2250 mMNaClNaCl1
31 mMEDTAEDTA1
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 FIELD / 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: 152123 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial model for chains C and D was generated using AlphaFold2
Atomic model building

3D fitting-ID: 1 / Accession code: 1JEY / Initial refinement model-ID: 1 / PDB-ID: 1JEY

1jey

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314033
ELECTRON MICROSCOPYf_angle_d0.47318948
ELECTRON MICROSCOPYf_dihedral_angle_d4.0831837
ELECTRON MICROSCOPYf_chiral_restr0.0412131
ELECTRON MICROSCOPYf_plane_restr0.0042432

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