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- PDB-7zvt: CryoEM structure of Ku heterodimer bound to DNA -

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Basic information

Entry
Database: PDB / ID: 7zvt
TitleCryoEM structure of Ku heterodimer bound to DNA
Components
  • DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')
  • DNA (5'-D(P*TP*CP*CP*CP*TP*CP*TP*AP*GP*AP*TP*AP*TP*C)-3')
  • X-ray repair cross-complementing protein 5
  • X-ray repair cross-complementing protein 6
KeywordsDNA BINDING PROTEIN / NHEJ / Ku70 / Ku80 / DNA damage
Function / homology
Function and homology information


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 complex / DNA-dependent protein kinase-DNA ligase 4 complex / cellular response to X-ray / nonhomologous end joining complex / regulation of smooth muscle cell proliferation ...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 complex / DNA-dependent protein kinase-DNA ligase 4 complex / cellular response to X-ray / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / nuclear telomere cap complex / double-strand break repair via classical nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / regulation of telomere maintenance / recombinational repair / U3 snoRNA binding / protein localization to chromosome, telomeric region / cellular response to fatty acid / cellular hyperosmotic salinity response / positive regulation of neurogenesis / telomeric DNA binding / 2-LTR circle formation / hematopoietic stem cell proliferation / site of DNA damage / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / ATP-dependent activity, acting on DNA / positive regulation of protein kinase activity / hematopoietic stem cell differentiation / telomere maintenance via telomerase / DNA helicase activity / neurogenesis / telomere maintenance / activation of innate immune response / cyclin binding / cellular response to leukemia inhibitory factor / enzyme activator activity / Nonhomologous End-Joining (NHEJ) / small-subunit processome / cellular response to gamma radiation / protein-DNA complex / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / double-strand break repair via nonhomologous end joining / double-strand break repair / double-stranded DNA binding / scaffold protein binding / secretory granule lumen / DNA recombination / transcription regulator complex / ficolin-1-rich granule lumen / damaged DNA binding / chromosome, telomeric region / transcription cis-regulatory region binding / response to xenobiotic stimulus / ribonucleoprotein complex / innate immune response / negative regulation of DNA-templated transcription / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / protein-containing complex binding / positive regulation of DNA-templated transcription / nucleolus / positive regulation of transcription by RNA polymerase II / protein-containing complex / ATP hydrolysis activity / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / nucleus / membrane / plasma membrane / cytosol
Similarity search - Function
Ku70; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / 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; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / 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 beta-barrel domain / Ku70/Ku80 N-terminal alpha/beta 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 / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / DNA / DNA (> 10) / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsHardwick, S.W. / Kefala-Stavridi, A. / Chirgadze, D.Y. / Blundell, T.L. / Chaplin, A.K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z; 2016 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.
Authors: Antonia Kefala Stavridi / Amandine Gontier / Vincent Morin / Philippe Frit / Virginie Ropars / Nadia Barboule / Carine Racca / Sagun Jonchhe / Michael J Morten / Jessica Andreani / Alexey ...Authors: Antonia Kefala Stavridi / Amandine Gontier / Vincent Morin / Philippe Frit / Virginie Ropars / Nadia Barboule / Carine Racca / Sagun Jonchhe / Michael J Morten / Jessica Andreani / Alexey Rak / Pierre Legrand / Alexa Bourand-Plantefol / Steven W Hardwick / Dimitri Y Chirgadze / Paul Davey / Taiana Maia De Oliveira / Eli Rothenberg / Sebastien Britton / Patrick Calsou / Tom L Blundell / Paloma F Varela / Amanda K Chaplin / Jean-Baptiste Charbonnier /
Abstract: The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by ...The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.
History
DepositionMay 17, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 6, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA (5'-D(P*TP*CP*CP*CP*TP*CP*TP*AP*GP*AP*TP*AP*TP*C)-3')
D: DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')
A: X-ray repair cross-complementing protein 6
B: X-ray repair cross-complementing protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,5705
Polymers161,9104
Non-polymers6601
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22830 Å2
ΔGint-159 kcal/mol
Surface area49750 Å2

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Components

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


Mass: 4190.741 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: DNA chain DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')


Mass: 4962.240 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Protein X-ray repair cross-complementing protein 6 / 5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP- ...5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 1 / ATP-dependent DNA helicase II 70 kDa subunit / CTC box-binding factor 75 kDa subunit / CTCBF / DNA repair protein XRCC6 / Lupus Ku autoantigen protein p70 / Ku70 / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 6


Mass: 69945.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P12956, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases
#4: 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 / 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: 82812.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#5: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
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: Ku70/80 heterodimer bound to DNA / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 8
SpecimenConc.: 2 mg/ml / Embedding 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 magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.25 sec. / Electron dose: 44.39 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6924

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2143882
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 276013 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.85 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00349078
ELECTRON MICROSCOPYf_angle_d0.528512378
ELECTRON MICROSCOPYf_chiral_restr0.03951388
ELECTRON MICROSCOPYf_plane_restr0.00311478
ELECTRON MICROSCOPYf_dihedral_angle_d17.01641391

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