+Open data
-Basic information
Entry | Database: PDB / ID: 8bot | ||||||
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Title | Cryo-EM structure of NHEJ supercomplex(trimer) | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / NHEJ / DNA-PK / DNA-PKcs / Ku70 / Ku80 / XLF / DNA repair | ||||||
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 / positive regulation of catalytic activity / B cell lineage commitment / double-strand break repair via alternative nonhomologous end joining / 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 / positive regulation of telomerase 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 / Nonhomologous End-Joining (NHEJ) / small-subunit processome / 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: 7.76 Å | ||||||
Authors | Hardwick, S.W. / Chaplin, A.K. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Structure / Year: 2023 Title: Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ. Authors: Steven W Hardwick / Antonia Kefala Stavridi / Dimitri Y Chirgadze / Taiana Maia De Oliveira / Jean-Baptiste Charbonnier / Virginie Ropars / Katheryn Meek / Tom L Blundell / Amanda K Chaplin / Abstract: The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ...The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8bot.cif.gz | 2.8 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8bot.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8bot.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8bot_validation.pdf.gz | 1.9 MB | Display | wwPDB validaton report |
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Full document | 8bot_full_validation.pdf.gz | 2.6 MB | Display | |
Data in XML | 8bot_validation.xml.gz | 482.8 KB | Display | |
Data in CIF | 8bot_validation.cif.gz | 734.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bo/8bot ftp://data.pdbj.org/pub/pdb/validation_reports/bo/8bot | HTTPS FTP |
-Related structure data
Related structure data | 16145MC 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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1 |
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-Components
-Protein , 4 types, 13 molecules KLNOMPQRXYFAS
#1: 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 #2: 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) #3: Protein | Mass: 33372.234 Da / Num. of mol.: 4 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 #4: Protein | Mass: 469673.219 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) References: UniProt: P78527, non-specific serine/threonine protein kinase |
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-X-ray repair cross-complementing protein ... , 2 types, 6 molecules GBTHCU
#5: Protein | Mass: 69945.039 Da / Num. of mol.: 3 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 #6: Protein | Mass: 82812.438 Da / Num. of mol.: 3 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 |
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-DNA chain , 4 types, 6 molecules IVJWDE
#7: DNA chain | Mass: 8619.629 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) #8: DNA chain | Mass: 8350.414 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) #9: DNA chain | | Mass: 7367.843 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) #10: DNA chain | | Mass: 7402.821 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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-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: NHEJ supercomplex trimer / Type: ORGANELLE OR CELLULAR COMPONENT Details: Trimeric complex of DNA-PK, DNA ligase 4, XRCC4, XLF Entity ID: all / Source: NATURAL |
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Molecular weight | Value: 2.1 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Buffer solution | pH: 8 |
Specimen | Conc.: 2 mg/ml / 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: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 46.8 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: 749185 | ||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 7.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9114 / Symmetry type: POINT | ||||||||||||||||||||||||||||||
Atomic model building | B value: 639 | ||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 636.65 Å2 | ||||||||||||||||||||||||||||||
Refine LS restraints |
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