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- PDB-5y3r: Cryo-EM structure of Human DNA-PK Holoenzyme -

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Entry
Database: PDB / ID: 5y3r
TitleCryo-EM structure of Human DNA-PK Holoenzyme
Components
  • (X-ray repair cross-complementing protein ...) x 2
  • DNA (34-MER)
  • DNA (36-MER)
  • DNA-dependent protein kinase catalytic subunit
  • PRKDC-Helix
KeywordsDNA BINDING PROTEIN / Cryo-EM structure / DNA-PK / DNAPKcs / activation / NHEJ
Function / homology
Function and homology information


positive regulation of platelet formation / Ku70:Ku80 complex / negative regulation of t-circle formation / T cell receptor V(D)J recombination / DNA end binding / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / DNA-dependent protein kinase complex ...positive regulation of platelet formation / Ku70:Ku80 complex / negative regulation of t-circle formation / T cell receptor V(D)J recombination / DNA end binding / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / DNA-dependent protein kinase complex / histone H2AXS139 kinase activity / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination / nonhomologous end joining complex / immature B cell differentiation / regulation of smooth muscle cell proliferation / cellular response to X-ray / nuclear telomere cap complex / double-strand break repair via alternative nonhomologous end joining / regulation of epithelial cell proliferation / double-strand break repair via classical nonhomologous end joining / telomere capping / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / positive regulation of neurogenesis / regulation of hematopoietic stem cell differentiation / recombinational repair / regulation of telomere maintenance / U3 snoRNA binding / protein localization to chromosome, telomeric region / maturation of 5.8S rRNA / T cell lineage commitment / cellular hyperosmotic salinity response / negative regulation of cGAS/STING signaling pathway / positive regulation of double-strand break repair via nonhomologous end joining / 2-LTR circle formation / B cell lineage commitment / telomeric DNA binding / hematopoietic stem cell proliferation / negative regulation of protein phosphorylation / positive regulation of protein kinase activity / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / site of DNA damage / peptidyl-threonine phosphorylation / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / ATP-dependent activity, acting on DNA / ectopic germ cell programmed cell death / somitogenesis / telomere maintenance via telomerase / mitotic G1 DNA damage checkpoint signaling / neurogenesis / telomere maintenance / activation of innate immune response / DNA helicase activity / cyclin binding / positive regulation of erythrocyte differentiation / positive regulation of translation / cellular response to leukemia inhibitory factor / response to gamma radiation / small-subunit processome / enzyme activator activity / Nonhomologous End-Joining (NHEJ) / cellular response to gamma radiation / protein-DNA complex / regulation of circadian rhythm / brain development / protein destabilization / peptidyl-serine phosphorylation / double-strand break repair via nonhomologous end joining / protein modification process / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to insulin stimulus / intrinsic apoptotic signaling pathway in response to DNA damage / rhythmic process / T cell differentiation in thymus / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / heart development / double-stranded DNA binding / scaffold protein binding / secretory granule lumen / DNA recombination / transcription regulator complex / ficolin-1-rich granule lumen / damaged DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / chromosome, telomeric region / protein phosphorylation / non-specific serine/threonine protein kinase / protein kinase activity / transcription cis-regulatory region binding / positive regulation of apoptotic process / ribonucleoprotein complex / protein domain specific binding / innate immune response / protein serine kinase activity / negative regulation of DNA-templated transcription / protein serine/threonine kinase activity / ubiquitin protein ligase binding
Similarity search - Function
Ku70, bridge and pillars domain superfamily / : / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal ...Ku70, bridge and pillars domain superfamily / : / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC1/2 / NUC194 / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 N-terminal alpha/beta domain / Ku70/Ku80 beta-barrel domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / Armadillo-like helical / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5 / DNA-dependent protein kinase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsYin, X. / Liu, M. / Tian, Y. / Wang, J. / Xu, Y.
Funding support China, 4items
OrganizationGrant numberCountry
Ministry of Science and Technology of China2016YFA0500700 China
Strategic Priority Research Program of the Chinese Academy of SciencesXDB08000000 China
National Natural Science Foundation of ChinaU1432242,31425008,91419301 China
Program of Shanghai Subject Chief Scientist14XD1400500 China
CitationJournal: Cell Res / Year: 2017
Title: Cryo-EM structure of human DNA-PK holoenzyme.
Authors: Xiaotong Yin / Mengjie Liu / Yuan Tian / Jiawei Wang / Yanhui Xu /
Abstract: DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical ...DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal α-solenoid (∼2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ∼30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.
History
DepositionJul 29, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 6, 2017Provider: repository / Type: Initial release
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Revision 1.0Sep 6, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Nov 15, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 13, 2018Group: Data collection / Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_host_org_cell_line / _entity_src_gen.pdbx_host_org_strain
Revision 1.3Oct 9, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.4Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.5Jul 2, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: X-ray repair cross-complementing protein 6
B: X-ray repair cross-complementing protein 5
K: PRKDC-Helix
D: DNA (34-MER)
E: DNA (36-MER)
C: DNA-dependent protein kinase catalytic subunit


Theoretical massNumber of molelcules
Total (without water)610,1076
Polymers610,1076
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area27230 Å2
ΔGint-158 kcal/mol
Surface area214470 Å2

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Components

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X-ray repair cross-complementing protein ... , 2 types, 2 molecules AB

#1: Protein X-ray repair cross-complementing protein 6 / 5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP- ...5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 1 / ATP-dependent DNA helicase II 70 kDa subunit / CTC box-binding factor 75 kDa subunit / CTCBF / DNA repair protein XRCC6 / Lupus Ku autoantigen protein p70 / Ku70 / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 6


Mass: 57619.352 Da / Num. of mol.: 1 / Fragment: UNP residues 34-534
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1 / Production host: Homo sapiens (human)
References: UniProt: P12956, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases
#2: Protein X-ray repair cross-complementing protein 5 / 86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase ...86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase II 80 kDa subunit / CTC box-binding factor 85 kDa subunit / CTCBF / DNA repair protein XRCC5 / Ku80 / Ku86 / Lupus Ku autoantigen protein p86 / Nuclear factor IV / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)


Mass: 60972.969 Da / Num. of mol.: 1 / Fragment: UNP residues 6-541
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Production host: Homo sapiens (human)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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DNA chain , 2 types, 2 molecules DE

#4: DNA chain DNA (34-MER)


Mass: 10502.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: DNA chain DNA (36-MER)


Mass: 11042.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Protein/peptide / Protein , 2 types, 2 molecules KC

#3: Protein/peptide PRKDC-Helix


Mass: 1084.182 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#6: Protein DNA-dependent protein kinase catalytic subunit / DNA-PKcs / DNPK1 / p460


Mass: 468885.344 Da / Num. of mol.: 1 / Fragment: UNP residues 10-4128
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKDC, HYRC, HYRC1 / Cell line (production host): HEK293F / Organ (production host): KIDNEY / Production host: Homo sapiens (human)
References: UniProt: P78527, non-specific serine/threonine protein kinase

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Details

Has protein modificationN

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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: PRKDC-Helix / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 18000 X / Nominal defocus max: 4700 nm / Nominal defocus min: 1700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3496
Image scansMovie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155 / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
2CTFFIND33CTF correction
3RELION2final Euler assignment
4RELION2classification
5RELION23D reconstruction
6PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53451 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01139702
ELECTRON MICROSCOPYf_angle_d1.553937
ELECTRON MICROSCOPYf_dihedral_angle_d12.27424059
ELECTRON MICROSCOPYf_chiral_restr0.0696075
ELECTRON MICROSCOPYf_plane_restr0.0096647

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