+Open data
-Basic information
Entry | Database: PDB / ID: 8bhy | ||||||
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Title | DNA-PK Ku80 mediated dimer bound to PAXX and XLF | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / DNA-PK / DNA-PKcs / Ku70 / Ku80 / PAXX / NHEJ / XLF | ||||||
Function / homology | Function and homology information DNA ligation involved in DNA recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / DNA ligase IV complex / DNA ligation involved in DNA repair / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / negative regulation of t-circle formation ...DNA ligation involved in DNA recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / DNA ligase IV complex / DNA ligation involved in DNA repair / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / negative regulation of t-circle formation / positive regulation of platelet formation / DNA ligase (ATP) / DNA end binding / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA ligase (ATP) activity / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / single strand break repair / cellular response to X-ray / immunoglobulin V(D)J recombination / nonhomologous end joining complex / DNA ligation / regulation of smooth muscle cell proliferation / V(D)J recombination / nuclear telomere cap complex / Cytosolic sensors of pathogen-associated DNA / double-strand break repair via classical nonhomologous end joining / isotype switching / protein localization to site of double-strand break / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / recombinational repair / regulation of telomere maintenance / regulation of hematopoietic stem cell differentiation / U3 snoRNA binding / positive regulation of neurogenesis / cellular response to fatty acid / nucleotide-excision repair, DNA gap filling / hematopoietic stem cell proliferation / protein localization to chromosome, telomeric region / cellular hyperosmotic salinity response / T cell lineage commitment / response to ionizing radiation / negative regulation of cGAS/STING signaling pathway / DNA biosynthetic process / cellular response to lithium ion / telomeric DNA binding / maturation of 5.8S rRNA / double-strand break repair via alternative nonhomologous end joining / positive regulation of catalytic activity / B cell lineage commitment / 2-LTR circle formation / positive regulation of double-strand break repair via nonhomologous end joining / ligase activity / site of DNA damage / somatic stem cell population maintenance / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / T cell differentiation / response to X-ray / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / chromosome organization / positive regulation of protein kinase activity / ectopic germ cell programmed cell death / ATP-dependent activity, acting on DNA / somitogenesis / SUMOylation of DNA damage response and repair proteins / DNA polymerase binding / condensed chromosome / DNA helicase activity / : / enzyme activator activity / mitotic G1 DNA damage checkpoint signaling / positive regulation of telomere maintenance via telomerase / activation of innate immune response / telomere maintenance / cyclin binding / B cell differentiation / neurogenesis / positive regulation of erythrocyte differentiation / negative regulation of protein phosphorylation / stem cell proliferation / cellular response to leukemia inhibitory factor / central nervous system development / positive regulation of translation / cellular response to ionizing radiation / response to gamma radiation / protein-DNA complex / small-subunit processome / Nonhomologous End-Joining (NHEJ) / peptidyl-threonine phosphorylation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein destabilization Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.33 Å | ||||||
Authors | Hardwick, S.W. / Chaplin, A.K. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Sci Adv / Year: 2023 Title: PAXX binding to the NHEJ machinery explains functional redundancy with XLF. Authors: Murielle Seif-El-Dahan / Antonia Kefala-Stavridi / Philippe Frit / Steven W Hardwick / Dima Y Chirgadze / Taiana Maia De Oliviera / Jessica Andreani / Sébastien Britton / Nadia Barboule / ...Authors: Murielle Seif-El-Dahan / Antonia Kefala-Stavridi / Philippe Frit / Steven W Hardwick / Dima Y Chirgadze / Taiana Maia De Oliviera / Jessica Andreani / Sébastien Britton / Nadia Barboule / Madeleine Bossaert / Arun Prasad Pandurangan / Katheryn Meek / Tom L Blundell / Virginie Ropars / Patrick Calsou / Jean-Baptiste Charbonnier / Amanda K Chaplin / Abstract: Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX ...Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8bhy.cif.gz | 2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8bhy.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8bhy.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8bhy_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8bhy_full_validation.pdf.gz | 2 MB | Display | |
Data in XML | 8bhy_validation.xml.gz | 315.8 KB | Display | |
Data in CIF | 8bhy_validation.cif.gz | 485.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bh/8bhy ftp://data.pdbj.org/pub/pdb/validation_reports/bh/8bhy | HTTPS FTP |
-Related structure data
Related structure data | 16074MC 7zwaC 7zygC 8ascC 8bh3C 8bhvC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
-Protein , 5 types, 12 molecules ASDMGHPQIRfm
#1: Protein | Mass: 469673.219 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) References: UniProt: P78527, non-specific serine/threonine protein kinase #4: Protein | Mass: 21663.498 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: PAXX, C9orf142, XLS / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9BUH6 #5: Protein | Mass: 38337.703 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q13426 #6: Protein | Mass: 104124.953 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LIG4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P49917, DNA ligase (ATP) #11: Protein | Mass: 33372.234 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: NHEJ1, XLF / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9H9Q4 |
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-X-ray repair cross-complementing protein ... , 2 types, 4 molecules BTCL
#2: Protein | Mass: 69945.039 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1 / Production host: Spodoptera frugiperda (fall armyworm) 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 #3: Protein | Mass: 82812.438 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement |
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-DNA chain , 4 types, 4 molecules deij
#7: DNA chain | Mass: 7733.016 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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#8: DNA chain | Mass: 8290.423 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
#9: DNA chain | Mass: 7961.218 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
#10: DNA chain | Mass: 7443.834 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: DNA-PK Ku80 mediated dimer bound to PAXX and XLF / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 1.6 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 48.03 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 343564 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 5.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30390 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 695.61 Å2 | ||||||||||||||||||||||||
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