+Open data
-Basic information
Entry | Database: PDB / ID: 7otw | ||||||
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Title | DNA-PKcs in complex with AZD7648 | ||||||
Components | DNA-dependent protein kinase catalytic subunit,DNA-PKcs | ||||||
Keywords | DNA BINDING PROTEIN / complex / inhibitor / DNA repair | ||||||
Function / homology | Function and homology information MHC class II antigen presentation / Neutrophil degranulation / entry into host cell by a symbiont-containing vacuole / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / protein autoprocessing / transport vesicle / protein processing / aspartic-type endopeptidase activity / proteolysis Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å | ||||||
Authors | Liang, S. / Thomas, S.E. / Blundell, T.L. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Nature / Year: 2022 Title: Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs. Authors: Shikang Liang / Sherine E Thomas / Amanda K Chaplin / Steven W Hardwick / Dimitri Y Chirgadze / Tom L Blundell / Abstract: The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in ...The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5'-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7otw.cif.gz | 647 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7otw.ent.gz | 520 KB | Display | PDB format |
PDBx/mmJSON format | 7otw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7otw_validation.pdf.gz | 982.6 KB | Display | wwPDB validaton report |
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Full document | 7otw_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 7otw_validation.xml.gz | 95.4 KB | Display | |
Data in CIF | 7otw_validation.cif.gz | 148.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ot/7otw ftp://data.pdbj.org/pub/pdb/validation_reports/ot/7otw | HTTPS FTP |
-Related structure data
Related structure data | 13068MC 7otmC 7otpC 7otvC 7otyC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 471375.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) References: UniProt: P78527, non-specific serine/threonine protein kinase |
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#2: Chemical | ChemComp-MBW / |
Has ligand of interest | Y |
-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: DNA-PKcs in complex with AZD7648 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL |
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Source (natural) | Organism: Homo sapiens (human) |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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 FIELD |
Image recording | Electron dose: 46.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164192 / Symmetry type: POINT | ||||||||||||||||||||||||
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