|Entry||Database: EMDB / ID: EMD-4425|
|Title||Cryo-EM structure of DNA-PKcs|
|Sample||DNA-dependent protein kinase catalytic subunit|
|Biological species||Homo (humans)|
|Method||single particle reconstruction / cryo EM / Resolution: 5.7 Å|
|Authors||Wu Q / Blundell TL|
|Funding support|| United Kingdom, 1 items |
|Citation||Journal: Prog. Biophys. Mol. Biol. / Year: 2019|
Title: Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways.
Authors: Qian Wu / Shikang Liang / Takashi Ochi / Dimitri Y Chirgadze / Juha T Huiskonen / Tom L Blundell /
Abstract: DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End ...DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End Joining repairs DNA double-strand breaks directly without a template and so can take place at any point in the cell cycle. Ku70/80 heterodimers rapidly assemble around broken DNA ends, allowing DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to be recruited and facilitating synapsis of broken DNA ends. This then provides a stage for end-processing and ligation. Here we review progress leading in 2017 to the medium resolution X-ray structure of DNA-PKcs, a single polypeptide chain of 4128 amino acids. This was followed quickly by chain tracing of cryo-EM structures of DNA-PKcs in complex with Ku and DNA. We discuss how combination of structural information from X-ray and cryo-EM studies can produce a working model for complex multicomponent molecular assemblies such as those found in DNA-double-strand-break repair.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_4425.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.43 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire DNA-dependent protein kinase catalytic subunit
|Entire||Name: DNA-dependent protein kinase catalytic subunit / Number of components: 2|
-Component #1: cellular-component, DNA-dependent protein kinase catalytic subunit
|Cellular-component||Name: DNA-dependent protein kinase catalytic subunit / Recombinant expression: No|
|Source||Species: Homo (humans)|
-Component #2: protein, DNA-dependent protein kinase catalytic subunit
|Protein||Name: DNA-dependent protein kinase catalytic subunit / Recombinant expression: No|
|Source||Species: Homo (humans)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.2 mg/mL|
Buffer solution: 20 mM Hepes pH 7.6, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
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