+Open data
-Basic information
Entry | Database: PDB / ID: 7nfe | ||||||
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Title | Cryo-EM structure of NHEJ super-complex (monomer) | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / NHEJ / DNA-PKcs / Ku70/80 / XLF / XRCC4 / DNA-LigaseIV | ||||||
Function / homology | Function and homology information DNA ligation involved in DNA recombination / T cell receptor V(D)J recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / pro-B cell differentiation / DNA ligase IV complex / DNA ligation involved in DNA repair / positive regulation of lymphocyte differentiation / small-subunit processome assembly ...DNA ligation involved in DNA recombination / T cell receptor V(D)J recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / pro-B cell differentiation / DNA ligase IV complex / DNA ligation involved in DNA repair / positive regulation of lymphocyte differentiation / small-subunit processome assembly / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / DNA-dependent protein kinase complex / immunoglobulin V(D)J recombination / negative regulation of t-circle formation / DNA end binding / DNA ligase (ATP) / DNA-dependent protein kinase-DNA ligase 4 complex / MHC class II antigen presentation / nonhomologous end joining complex / DNA ligase (ATP) activity / cellular response to X-ray / regulation of smooth muscle cell proliferation / single strand break repair / Cytosolic sensors of pathogen-associated DNA / Neutrophil degranulation / DNA ligation / V(D)J recombination / IRF3-mediated induction of type I IFN / nuclear telomere cap complex / nucleotide-excision repair, DNA gap filling / isotype switching / double-strand break repair via classical nonhomologous end joining / protein localization to site of double-strand break / entry into host cell by a symbiont-containing vacuole / positive regulation of catalytic activity / U3 snoRNA binding / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / positive regulation of neurogenesis / cellular response to fatty acid / cellular hyperosmotic salinity response / hematopoietic stem cell proliferation / response to ionizing radiation / cellular response to lithium ion / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / DNA biosynthetic process / telomeric DNA binding / 2-LTR circle formation / ligase activity / : / somatic stem cell population maintenance / site of DNA damage / protein autoprocessing / T cell differentiation / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / response to X-ray / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / chromosome organization / ATP-dependent activity, acting on DNA / SUMOylation of DNA damage response and repair proteins / DNA polymerase binding / activation of innate immune response / condensed chromosome / enzyme activator activity / transport vesicle / positive regulation of telomere maintenance via telomerase / telomere maintenance / DNA helicase activity / neurogenesis / B cell differentiation / cyclin binding / protein-DNA complex / stem cell proliferation / response to gamma radiation / cellular response to leukemia inhibitory factor / central nervous system development / small-subunit processome / cellular response to ionizing radiation / Nonhomologous End-Joining (NHEJ) / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to gamma radiation / fibrillar center / protein processing / double-strand break repair via nonhomologous end joining / establishment of integrated proviral latency / positive regulation of fibroblast proliferation / double-strand break repair / site of double-strand break / T cell differentiation in thymus / double-stranded DNA binding / scaffold protein binding / fibroblast proliferation / secretory granule lumen / in utero embryonic development / DNA recombination Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.29 Å | ||||||
Authors | Chaplin, A.K. / Hardwick, S.W. / Kefala Stavridi, A. / Chirgadze, D.Y. / Blundell, T.L. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Mol Cell / Year: 2021 Title: Cryo-EM of NHEJ supercomplexes provides insights into DNA repair. Authors: Amanda K Chaplin / Steven W Hardwick / Antonia Kefala Stavridi / Christopher J Buehl / Noah J Goff / Virginie Ropars / Shikang Liang / Taiana Maia De Oliveira / Dimitri Y Chirgadze / ...Authors: Amanda K Chaplin / Steven W Hardwick / Antonia Kefala Stavridi / Christopher J Buehl / Noah J Goff / Virginie Ropars / Shikang Liang / Taiana Maia De Oliveira / Dimitri Y Chirgadze / Katheryn Meek / Jean-Baptiste Charbonnier / Tom L Blundell / Abstract: Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or ...Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7nfe.cif.gz | 1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7nfe.ent.gz | 808.7 KB | Display | PDB format |
PDBx/mmJSON format | 7nfe.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7nfe_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7nfe_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 7nfe_validation.xml.gz | 160.4 KB | Display | |
Data in CIF | 7nfe_validation.cif.gz | 244.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nf/7nfe ftp://data.pdbj.org/pub/pdb/validation_reports/nf/7nfe | HTTPS FTP |
-Related structure data
Related structure data | 12301MC 7nfcC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 4 types, 6 molecules AFGHIJ
#1: Protein | Mass: 472056.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA References: UniProt: P78527, non-specific serine/threonine protein kinase | ||||
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#4: 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 #5: Protein | Mass: 38337.703 Da / Num. of mol.: 2 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.: 1 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) |
-X-ray repair cross-complementing protein ... , 2 types, 2 molecules BC
#2: Protein | 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 |
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#3: Protein | 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 |
-DNA chain , 2 types, 2 molecules DE
#7: DNA chain | Mass: 7367.843 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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#8: DNA chain | Mass: 7402.821 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
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Molecular weight | Value: 0.8 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.4 | ||||||||||||||||||||||||||||||||||||
Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||
Vitrification | 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 / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.3 sec. / Electron dose: 46.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
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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: 4.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45943 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 358.24 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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