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- EMDB-1208: 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-1208 | |||||||||
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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 structure of the DNA-bound DNAPKcs/Ku70/ku80 complex using cryoEM | |||||||||
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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 / cryo EM / Resolution: 32.0 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 | ![]() | 990.6 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.7 KB 11.7 KB | Display Display | ![]() |
Images | ![]() | 61.1 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 structure of the DNA-bound DNAPKcs/Ku70/ku80 complex using cryoEM | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : Macromolecular Complex containing DNA, DNAPKcs, Ku70 and Ku80
Entire | Name: Macromolecular Complex containing DNA, DNAPKcs, Ku70 and Ku80 |
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Components |
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-Supramolecule #1000: Macromolecular Complex containing DNA, DNAPKcs, Ku70 and Ku80
Supramolecule | Name: Macromolecular Complex containing DNA, DNAPKcs, Ku70 and Ku80 type: sample / ID: 1000 Oligomeric state: One molecule of DNAPKcs and one Ku70-Ku80 dimer bound to one DNA molecule Number unique components: 4 |
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Molecular weight | Theoretical: 650 KDa |
-Macromolecule #1: DNA-PKcs
Macromolecule | Name: DNA-PKcs / type: protein_or_peptide / ID: 1 / Number of copies: 1 / 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: 1 / 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: 1 / 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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-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 / Details: 20 mM Hepes, 200 mM KCl, 0.1 mM DTT, 0.5 mM EDTA |
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Grid | Details: 400 mesh carbon coated Rhodium-Copper grids |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home made plunger / Method: Blot for a few seconds before plunging |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 90 K |
Alignment procedure | Legacy - Astigmatism: FFT with ssCCD |
Details | Microscope model: TECNAI G2 200 kV, FEI. |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 32 / Details: Scanner: MINOLTA Dimage Scan Multi Pro / Bits/pixel: 16 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: side entry liquid-nitrogen cooled cryo specimen holder. Eucentric Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
CTF correction | Details: Each partcile using CTFIND3 and EMAN. |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 4189 |