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- EMDB-1210: Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 compl... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-1210 | |||||||||
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Title | Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. | |||||||||
![]() | 3D reconstruction of dimers containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes, obtained from negatively stained samples | |||||||||
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Function / homology | : / Ku70 / Phosphatidylinositol 3-/4-kinase, catalytic domain / double-strand break repair via nonhomologous end joining / double-strand break repair / protein kinase activity![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 33.0 Å | |||||||||
![]() | Spagnolo L / Rivera-Calzada A / Pearl LH / Llorca O | |||||||||
![]() | ![]() Title: Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Authors: Laura Spagnolo / Angel Rivera-Calzada / Laurence H Pearl / Oscar Llorca / ![]() Abstract: DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by ...DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 209.4 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.8 KB 11.8 KB | Display Display | ![]() |
Images | ![]() | 38.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of dimers containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes, obtained from negatively stained samples | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.4 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : dimer containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes
Entire | Name: dimer containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes |
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Components |
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-Supramolecule #1000: dimer containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes
Supramolecule | Name: dimer containing two DNA-bound DNAPKcs-Ku70-Ku80 complexes type: sample / ID: 1000 Oligomeric state: Two DNA-PKcs molecules bind two Ku70 and two Ku80 molecules on two DNA molecules Number unique components: 4 |
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Molecular weight | Theoretical: 1.3 MDa |
-Macromolecule #1: DNA-PKcs
Macromolecule | Name: DNA-PKcs / type: protein_or_peptide / ID: 1 Name.synonym: DNA-dependent Protein Kinase catalytic subunit Number of copies: 2 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 470 KDa |
Recombinant expression | Organism: HeLa cells |
Sequence | GO: protein kinase activity InterPro: Phosphatidylinositol 3-/4-kinase, catalytic domain |
-Macromolecule #2: Ku70
Macromolecule | Name: Ku70 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 70 KDa |
Recombinant expression | Organism: HeLa cells |
Sequence | GO: double-strand break repair via nonhomologous end joining InterPro: Ku70 |
-Macromolecule #3: ku80
Macromolecule | Name: ku80 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 85 KDa |
Recombinant expression | Organism: HeLa cells |
Sequence | GO: double-strand break repair / InterPro: INTERPRO: IPR011210 |
-Macromolecule #4: DNA
Macromolecule | Name: DNA / type: dna / ID: 4 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Sequence | String: ACGCGTGCGG CCATAATAAT AGTTTTTAGT TTATTGGGCG CG |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Details: 20 mM HEPES pH 7.5, 0.5 mM DTT, 0.25 mM EDTA, 0.0005% beta-octylglucoside, 25% glycerol, 100 mM NaCl |
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Staining | Type: NEGATIVE Details: The sample was applied to carbon-coated grids after glow-discharge and negatively stained with 1% uranyl acetate |
Grid | Details: 400 mesh Rhodium-copper grids |
Vitrification | Cryogen name: NONE |
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Electron microscopy
Microscope | JEOL 1230 |
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Alignment procedure | Legacy - Astigmatism: correction with FFT and CCD camera |
Details | Microscope: JEOL 1230 operated at 100 Kv. Specimen holder, JEOL type M: 207EM-11020. |
Image recording | Category: FILM / Film or detector model: KODAK 4489 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 84 / Details: Scanner: MINOLTA Dimage Scan Multi Pro scanner / Bits/pixel: 16 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm / Nominal magnification: 50000 |
Sample stage | Specimen holder: JEOL type M / Specimen holder model: OTHER |
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Image processing
Details | Structure solved applying 2-fold rotational symmetry |
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Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 33.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN Details: The structure was refined applying 2-fold rotational symmetry Number images used: 2151 |