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- PDB-6zh2: Cryo-EM structure of DNA-PKcs (State 1) -

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Basic information

Entry
Database: PDB / ID: 6zh2
TitleCryo-EM structure of DNA-PKcs (State 1)
ComponentsDNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs
KeywordsDNA BINDING PROTEIN / Kinase / DNA-PKcs / NHEJ / DNA-repair / DNA-PK
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
DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / Pepsin-like domain / FATC domain / FAT domain / Phosphatidylinositol 3- and 4-kinase / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. ...DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / Pepsin-like domain / FATC domain / FAT domain / Phosphatidylinositol 3- and 4-kinase / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsChaplin, A.K. / Hardwick, S.W. / Chirgadze, D.Y. / Blundell, T.L.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z United Kingdom
Citation
Journal: Nat Struct Mol Biol / Year: 2021
Title: Dimers of DNA-PK create a stage for DNA double-strand break repair.
Authors: Amanda K Chaplin / Steven W Hardwick / Shikang Liang / Antonia Kefala Stavridi / Ales Hnizda / Lee R Cooper / Taiana Maia De Oliveira / Dimitri Y Chirgadze / Tom L Blundell /
Abstract: DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent ...DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.
#1: Journal: Nat.Struct.Mol.Biol. / Year: 2020
Title: Dimers of DNA-PK create a stage for DNA-double strand break repair
Authors: Chaplin, A.K. / Hardwick, S.W. / Liang, S. / Stavridi, A.K. / Hnizda, A. / Cooper, L.R. / De Oliveira, T.M. / Chirgadze, D.Y. / Blundell, T.L.
History
DepositionJun 20, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 4, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs


Theoretical massNumber of molelcules
Total (without water)472,0561
Polymers472,0561
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area161920 Å2
MethodPISA

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Components

#1: Protein DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs / DNA-PKcs / DNPK1 / p460


Mass: 472056.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered ...Details: UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily,UNK residues (X) are unknown elements of the DNA-PKcs molecule that have been numbered arbitrarily
Source: (natural) Homo sapiens (human) / Cell line: HELA
References: UniProt: P78527, non-specific serine/threonine protein kinase
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DNA-PKcs (state 1) / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.47 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 53.95 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameCategory
2EPUimage acquisition
4WarpCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13cryoSPARCmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 378084
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38575 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 142.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003329950
ELECTRON MICROSCOPYf_angle_d0.6540528
ELECTRON MICROSCOPYf_chiral_restr0.04114614
ELECTRON MICROSCOPYf_plane_restr0.00375164
ELECTRON MICROSCOPYf_dihedral_angle_d20.798311158

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