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- EMDB-1102: Three-dimensional structure and regulation of the DNA-dependent p... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-1102 | |||||||||
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Title | Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). | |||||||||
![]() | This is the cryoEM 3D reconstruction of DNA-PKcs kinase. | |||||||||
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Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 13.0 Å | |||||||||
![]() | Rivera-Calzada A / Maman JP / Spagnolo L / Pearl LH / Llorca O | |||||||||
![]() | ![]() Title: Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Authors: Angel Rivera-Calzada / Joseph D Maman / Laura Spagnolo / Laurence H Pearl / Oscar Llorca / ![]() Abstract: DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have ...DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have now generated an approximately 13 A three-dimensional map of DNA-PKcs, revealing the overall architecture and topology of the 4128 residue polypeptide chain and allowing location of domains. The highly conserved C-terminal PIKK catalytic domain forms a central structure from which FAT and FATC domains protrude. Conformational changes observed in these domains on DNA binding suggest that they transduce DNA-induced conformational changes to the catalytic core and regulate kinase activity. The N-terminal segments form long curved tubular-shaped domains based on helical repeats to create interacting surfaces required for macromolecular assembly. Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family. | |||||||||
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 | ![]() | 155 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.5 KB 10.5 KB | Display Display | ![]() |
Images | ![]() ![]() | 50.3 KB 263.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is the cryoEM 3D reconstruction of DNA-PKcs kinase. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 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 : Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
Entire | Name: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs |
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Components |
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-Supramolecule #1000: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
Supramolecule | Name: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1 |
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Molecular weight | Theoretical: 470 KDa / Method: From its sequence |
-Macromolecule #1: DNA-dependent protein kinase catalytic subunit
Macromolecule | Name: DNA-dependent protein kinase catalytic subunit / type: protein_or_peptide / ID: 1 / Name.synonym: DNA-PKcs / 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.2 mg/mL |
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Buffer | pH: 7.5 / Details: 50mM Tris-HCl, 50mM KCl, 1mM MgCl2. |
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 1 second before plunging |
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Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Min: 90 K / Max: 90 K / Average: 90 K |
Alignment procedure | Legacy - Astigmatism: objective astigmatism was corrected using the stigmator, CCD camera collected images and the Digital Micrograph software |
Details | Microscope model: TECNAI G2 200 kV, FEI. |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 43 / Details: Images were averaged to 14 microns / Bits/pixel: 8 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: side entry liquid-nitrogen cooled cryo specimen holder Specimen holder model: GATAN LIQUID NITROGEN |
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Image processing
CTF correction | Details: reverse phases for each particle |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: EMAN Details: CTF Corrected particles were subjected to 3D refinement as implemented in EMAN. The starting volume was generated by image classification of the whole data set into a few average images to ...Details: CTF Corrected particles were subjected to 3D refinement as implemented in EMAN. The starting volume was generated by image classification of the whole data set into a few average images to build a reconstruction using common lines. Number images used: 7000 |
Final angle assignment | Details: EMAN software criteria |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: SITUS |
Details | Protocol: Rigid Body. The domains were fitted using the COLORES command from SITUS |
Refinement | Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coeficient |