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- EMDB-14986: CryoEM structure of Ku heterodimer bound to DNA -

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Entry
Database: EMDB / ID: EMD-14986
TitleCryoEM structure of Ku heterodimer bound to DNA
Map dataKu hetero-dimer bound to DNA
Sample
  • Organelle or cellular component: Ku70/80 heterodimer bound to DNA
    • DNA: DNA (5'-D(P*TP*CP*CP*CP*TP*CP*TP*AP*GP*AP*TP*AP*TP*C)-3')
    • DNA: DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')
    • Protein or peptide: X-ray repair cross-complementing protein 6
    • Protein or peptide: X-ray repair cross-complementing protein 5
  • Ligand: INOSITOL HEXAKISPHOSPHATE
KeywordsNHEJ / Ku70 / Ku80 / DNA damage / DNA BINDING PROTEIN
Function / homology
Function and homology information


positive regulation of lymphocyte differentiation / small-subunit processome assembly / Ku70:Ku80 complex / DNA-dependent protein kinase complex / negative regulation of t-circle formation / DNA end binding / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / cellular response to X-ray / regulation of smooth muscle cell proliferation ...positive regulation of lymphocyte differentiation / small-subunit processome assembly / Ku70:Ku80 complex / DNA-dependent protein kinase complex / negative regulation of t-circle formation / DNA end binding / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / cellular response to X-ray / regulation of smooth muscle cell proliferation / Cytosolic sensors of pathogen-associated DNA / DNA ligation / IRF3-mediated induction of type I IFN / nuclear telomere cap complex / double-strand break repair via classical nonhomologous end joining / positive regulation of catalytic activity / U3 snoRNA binding / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / cellular response to fatty acid / positive regulation of neurogenesis / cellular hyperosmotic salinity response / hematopoietic stem cell proliferation / telomeric DNA binding / 2-LTR circle formation / : / site of DNA damage / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / ATP-dependent activity, acting on DNA / activation of innate immune response / enzyme activator activity / positive regulation of telomere maintenance via telomerase / DNA helicase activity / telomere maintenance / cyclin binding / neurogenesis / protein-DNA complex / cellular response to leukemia inhibitory factor / small-subunit processome / Nonhomologous End-Joining (NHEJ) / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to gamma radiation / 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 / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / nucleolus / positive regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / protein-containing complex / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / plasma membrane / cytosol
Similarity search - Function
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, 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 domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily
Similarity search - Domain/homology
X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsHardwick SW / Kefala-Stavridi A / Chirgadze DY / Blundell TL / Chaplin AK
Funding support United Kingdom, 1 items
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, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateDec 6, 2023-
Current statusDec 6, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14986.png

Downloads & links

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Map

FileDownload / File: emd_14986.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKu hetero-dimer bound to DNA
Projections & slices

Image control

Size
Brightness
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Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.65 Å/pix.
x 380 pix.
= 247. Å		0.65 Å/pix.
x 380 pix.
= 247. Å		0.65 Å/pix.
x 380 pix.
= 247. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.086
Minimum - Maximum-0.26406714 - 0.51470953
Average (Standard dev.)0.00024686786 (±0.012367518)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 246.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_14986_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14986_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Ku70/80 heterodimer bound to DNA

EntireName: Ku70/80 heterodimer bound to DNA
Components
  • Organelle or cellular component: Ku70/80 heterodimer bound to DNA
    • DNA: DNA (5'-D(P*TP*CP*CP*CP*TP*CP*TP*AP*GP*AP*TP*AP*TP*C)-3')
    • DNA: DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')
    • Protein or peptide: X-ray repair cross-complementing protein 6
    • Protein or peptide: X-ray repair cross-complementing protein 5
  • Ligand: INOSITOL HEXAKISPHOSPHATE

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Supramolecule #1: Ku70/80 heterodimer bound to DNA

SupramoleculeName: Ku70/80 heterodimer bound to DNA / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 150 KDa

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

MacromoleculeName: DNA (5'-D(P*TP*CP*CP*CP*TP*CP*TP*AP*GP*AP*TP*AP*TP*C)-3')
type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.190741 KDa
SequenceString:
(DT)(DC)(DC)(DC)(DT)(DC)(DT)(DA)(DG)(DA) (DT)(DA)(DT)(DC)

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Macromolecule #2: DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*AP*TP*AP*TP*CP*TP*AP*GP*AP*GP*GP*GP*AP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.96224 KDa
SequenceString:
(DC)(DG)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DG) (DA)(DG)(DG)(DG)(DA)(DT)

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Macromolecule #3: X-ray repair cross-complementing protein 6

MacromoleculeName: X-ray repair cross-complementing protein 6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.945039 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYG TEKDKNSVNF KNIYVLQELD NPGAKRILEL DQFKGQQGQK RFQDMMGHGS DYSLSEVLWV CANLFSDVQF K MSHKRIML ...String:
MSGWESYYKT EGDEEAEEEQ EENLEASGDY KYSGRDSLIF LVDASKAMFE SQSEDELTPF DMSIQCIQSV YISKIISSDR DLLAVVFYG TEKDKNSVNF KNIYVLQELD NPGAKRILEL DQFKGQQGQK RFQDMMGHGS DYSLSEVLWV CANLFSDVQF K MSHKRIML FTNEDNPHGN DSAKASRART KAGDLRDTGI FLDLMHLKKP GGFDISLFYR DIISIAEDED LRVHFEESSK LE DLLRKVR AKETRKRALS RLKLKLNKDI VISVGIYNLV QKALKPPPIK LYRETNEPVK TKTRTFNTST GGLLLPSDTK RSQ IYGSRQ IILEKEETEE LKRFDDPGLM LMGFKPLVLL KKHHYLRPSL FVYPEESLVI GSSTLFSALL IKCLEKEVAA LCRY TPRRN IPPYFVALVP QEEELDDQKI QVTPPGFQLV FLPFADDKRK MPFTEKIMAT PEQVGKMKAI VEKLRFTYRS DSFEN PVLQ QHFRNLEALA LDLMEPEQAV DLTLPKVEAM NKRLGSLVDE FKELVYPPDY NPEGKVTKRK HDNEGSGSKR PKVEYS EEE LKTHISKGTL GKFTVPMLKE ACRAYGLKSG LKKQELLEAL TKHFQD

UniProtKB: X-ray repair cross-complementing protein 6

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Macromolecule #4: X-ray repair cross-complementing protein 5

MacromoleculeName: X-ray repair cross-complementing protein 5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 82.812438 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFD LLEDIESKIQ PGSQQADFLD ALIVSMDVIQ HETIGKKFEK RHIEIFTDLS SRFSKSQLDI IIHSLKKCDI S LQFFLPFS ...String:
MVRSGNKAAV VLCMDVGFTM SNSIPGIESP FEQAKKVITM FVQRQVFAEN KDEIALVLFG TDGTDNPLSG GDQYQNITVH RHLMLPDFD LLEDIESKIQ PGSQQADFLD ALIVSMDVIQ HETIGKKFEK RHIEIFTDLS SRFSKSQLDI IIHSLKKCDI S LQFFLPFS LGKEDGSGDR GDGPFRLGGH GPSFPLKGIT EQQKEGLEIV KMVMISLEGE DGLDEIYSFS ESLRKLCVFK KI ERHSIHW PCRLTIGSNL SIRIAAYKSI LQERVKKTWT VVDAKTLKKE DIQKETVYCL NDDDETEVLK EDIIQGFRYG SDI VPFSKV DEEQMKYKSE GKCFSVLGFC KSSQVQRRFF MGNQVLKVFA ARDDEAAAVA LSSLIHALDD LDMVAIVRYA YDKR ANPQV GVAFPHIKHN YECLVYVQLP FMEDLRQYMF SSLKNSKKYA PTEAQLNAVD ALIDSMSLAK KDEKTDTLED LFPTT KIPN PRFQRLFQCL LHRALHPREP LPPIQQHIWN MLNPPAEVTT KSQIPLSKIK TLFPLIEAKK KDQVTAQEIF QDNHED GPT AKKLKTEQGG AHFSVSSLAE GSVTSVGSVN PAENFRVLVK QKKASFEEAS NQLINHIEQF LDTNETPYFM KSIDCIR AF REEAIKFSEE QRFNNFLKAL QEKVEIKQLN HFWEIVVQDG ITLITKEEAS GSSVTAEEAK KFLAPKDKPS GDTAAVFE E GGDVDDLLDM I

UniProtKB: X-ray repair cross-complementing protein 5

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Macromolecule #5: INOSITOL HEXAKISPHOSPHATE

MacromoleculeName: INOSITOL HEXAKISPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: IHP
Molecular weightTheoretical: 660.035 Da
Chemical component information

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6924 / Average exposure time: 1.25 sec. / Average electron dose: 44.39 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2143882
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1jey

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 276013
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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