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- EMDB-14955: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate -

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Entry
Database: EMDB / ID: EMD-14955
TitleCryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate
Map data
Sample
  • Complex: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate
    • Protein or peptide: X-ray repair cross-complementing protein 6
    • Protein or peptide: X-ray repair cross-complementing protein 5
  • Ligand: INOSITOL HEXAKISPHOSPHATEPhytic acid
KeywordsDNA repair / NHEJ / Ku 70/80 / DNA-PK / cancer / double-strand break / DNA binding protein
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 / DNA ligation ...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 / DNA ligation / nuclear telomere cap complex / regulation of smooth muscle cell proliferation / Cytosolic sensors of pathogen-associated DNA / double-strand break repair via classical nonhomologous end joining / IRF3-mediated induction of type I IFN / recombinational repair / regulation of telomere maintenance / U3 snoRNA binding / positive regulation of neurogenesis / protein localization to chromosome, telomeric region / cellular response to fatty acid / hematopoietic stem cell proliferation / cellular hyperosmotic salinity response / telomeric DNA binding / positive regulation of catalytic activity / 2-LTR circle formation / site of DNA damage / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / enzyme activator activity / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / ATP-dependent activity, acting on DNA / positive regulation of telomerase activity / positive regulation of telomere maintenance via telomerase / DNA helicase activity / telomere maintenance / activation of innate immune response / cellular response to leukemia inhibitory factor / small-subunit processome / neurogenesis / cyclin binding / protein-DNA complex / 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 / ficolin-1-rich granule lumen / transcription regulator complex / 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 / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / 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, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain ...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, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain / Ku70/Ku80 C-terminal arm / 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: 3.5 Å
AuthorsKefala Stavridi A / Chaplin AK / Blundell TL
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z 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 9, 2022-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateDec 6, 2023-
Current statusDec 6, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14955.png

Downloads & links

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Map

FileDownload / File: emd_14955.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 300 pix.
= 195.6 Å		0.65 Å/pix.
x 300 pix.
= 195.6 Å		0.65 Å/pix.
x 300 pix.
= 195.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0308
Minimum - Maximum-0.050727684 - 0.15665554
Average (Standard dev.)0.0012541697 (±0.0081452)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 195.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_14955_half_map_1.map
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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_14955_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate

EntireName: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate
Components
  • Complex: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate
    • Protein or peptide: X-ray repair cross-complementing protein 6
    • Protein or peptide: X-ray repair cross-complementing protein 5
  • Ligand: INOSITOL HEXAKISPHOSPHATEPhytic acid

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Supramolecule #1: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate

SupramoleculeName: Cryo-EM structure of Ku 70/80 bound to inositol hexakisphosphate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: X-ray repair cross-complementing protein 6

MacromoleculeName: X-ray repair cross-complementing protein 6 / type: protein_or_peptide / ID: 1 / 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: 73.890336 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNTIHHHHHH NTSGSGGGGG RLVPRGSMSE NLYFQGSMSG WESYYKTEGD EEAEEEQEEN LEASGDYKYS GRDSLIFLVD ASKAMFESQ SEDELTPFDM SIQCIQSVYI SKIISSDRDL LAVVFYGTEK DKNSVNFKNI YVLQELDNPG AKRILELDQF K GQQGQKRF ...String:
MNTIHHHHHH NTSGSGGGGG RLVPRGSMSE NLYFQGSMSG WESYYKTEGD EEAEEEQEEN LEASGDYKYS GRDSLIFLVD ASKAMFESQ SEDELTPFDM SIQCIQSVYI SKIISSDRDL LAVVFYGTEK DKNSVNFKNI YVLQELDNPG AKRILELDQF K GQQGQKRF QDMMGHGSDY SLSEVLWVCA NLFSDVQFKM SHKRIMLFTN EDNPHGNDSA KASRARTKAG DLRDTGIFLD LM HLKKPGG FDISLFYRDI ISIAEDEDLR VHFEESSKLE DLLRKVRAKE TRKRALSRLK LKLNKDIVIS VGIYNLVQKA LKP PPIKLY RETNEPVKTK TRTFNTSTGG LLLPSDTKRS QIYGSRQIIL EKEETEELKR FDDPGLMLMG FKPLVLLKKH HYLR PSLFV YPEESLVIGS STLFSALLIK CLEKEVAALC RYTPRRNIPP YFVALVPQEE ELDDQKIQVT PPGFQLVFLP FADDK RKMP FTEKIMATPE QVGKMKAIVE KLRFTYRSDS FENPVLQQHF RNLEALALDL MEPEQAVDLT LPKVEAMNKR LGSLVD EFK ELVYPPDYNP EGKVTKRKHD NEGSGSKRPK VEYSEEELKT HISKGTLGKF TVPMLKEACR AYGLKSGLKK QELLEAL TK HFQD

UniProtKB: X-ray repair cross-complementing protein 6

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Macromolecule #2: X-ray repair cross-complementing protein 5

MacromoleculeName: X-ray repair cross-complementing protein 5 / type: protein_or_peptide / ID: 2 / 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: Spodoptera frugiperda (fall armyworm)
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 #3: INOSITOL HEXAKISPHOSPHATE

MacromoleculeName: INOSITOL HEXAKISPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: IHP
Molecular weightTheoretical: 660.035 Da
Chemical component information

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE / Phytic acid

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.43 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1jey

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93175
FSC plot (resolution estimation)

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