+Open data
-Basic information
Entry | Database: PDB / ID: 8eza | |||||||||
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Title | NHEJ Long-range complex with PAXX | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / Complex / Kinase / NHEJ / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
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 end binding / DNA ligase (ATP) / 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 / protein localization to site of double-strand break / 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 / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / recombinational repair / regulation of telomere maintenance / nucleotide-excision repair, DNA gap filling / double-strand break repair via alternative nonhomologous end joining / regulation of hematopoietic stem cell differentiation / U3 snoRNA binding / positive regulation of neurogenesis / cellular response to fatty acid / hematopoietic stem cell proliferation / protein localization to chromosome, telomeric region / cellular hyperosmotic salinity response / maturation of 5.8S rRNA / T cell lineage commitment / response to ionizing radiation / negative regulation of cGAS/STING signaling pathway / telomeric DNA binding / DNA biosynthetic process / cellular response to lithium ion / B cell lineage commitment / positive regulation of catalytic activity / 2-LTR circle formation / positive regulation of double-strand break repair via nonhomologous end joining / site of DNA damage / somatic stem cell population maintenance / ligase activity / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / T cell differentiation / response to X-ray / 5'-deoxyribose-5-phosphate lyase activity / chromosome organization / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / ectopic germ cell programmed cell death / ATP-dependent activity, acting on DNA / enzyme activator activity / somitogenesis / SUMOylation of DNA damage response and repair proteins / DNA polymerase binding / condensed chromosome / positive regulation of telomerase activity / mitotic G1 DNA damage checkpoint signaling / positive regulation of telomere maintenance via telomerase / activation of innate immune response / DNA helicase activity / telomere maintenance / cyclin binding / neurogenesis / B cell differentiation / positive regulation of erythrocyte differentiation / negative regulation of protein phosphorylation / cellular response to leukemia inhibitory factor / central nervous system development / stem cell proliferation / positive regulation of translation / cellular response to ionizing radiation / small-subunit processome / protein-DNA complex / response to gamma radiation / 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: 4.39 Å | |||||||||
Authors | Chen, S. / He, Y. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Sci Adv / Year: 2023 Title: Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ. Authors: Siyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He / Abstract: DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). ...DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen ). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8eza.cif.gz | 2.1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8eza.ent.gz | 1.7 MB | Display | PDB format |
PDBx/mmJSON format | 8eza.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ez/8eza ftp://data.pdbj.org/pub/pdb/validation_reports/ez/8eza | HTTPS FTP |
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-Related structure data
Related structure data | 28732MC 8ez9C 8ezbC 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 , 5 types, 12 molecules STLCHIFGOPXY
#1: 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 (E. coli) / References: UniProt: Q9BUH6 #4: Protein | Mass: 469673.219 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA / References: UniProt: P78527 #8: 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 (E. coli) / References: UniProt: Q9H9Q4 #9: 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 (E. coli) / References: UniProt: Q13426 #10: 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 (E. coli) / References: UniProt: P49917, DNA ligase (ATP) |
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-X-ray repair cross-complementing protein ... , 2 types, 4 molecules JAKB
#2: Protein | Mass: 69945.039 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: unidentified baculovirus / References: UniProt: P12956 #3: Protein | Mass: 82812.438 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: unidentified baculovirus / References: UniProt: P13010 |
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-DNA chain , 2 types, 4 molecules MDNE
#6: DNA chain | Mass: 9510.159 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) #7: DNA chain | Mass: 9236.976 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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-Protein/peptide / Non-polymers , 2 types, 4 molecules RQ
#11: Chemical | #5: Protein/peptide | Mass: 1720.111 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA / References: non-specific serine/threonine protein kinase |
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-Details
Has ligand of interest | Y |
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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 Long-range complex with PAXX / Type: COMPLEX / Entity ID: #1-#7, #9-#10, #8 / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 1.75 MDa / Experimental value: NO |
Buffer solution | pH: 7.9 |
Specimen | Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 302 K |
-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 FIELDBright-field microscopy / Nominal magnification: 60000 X / Calibrated magnification: 60000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 4000 nm / Cs: 2 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K |
Image recording | Average exposure time: 4 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11006 |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1101255 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138252 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient |