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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


Ku70:Ku80 complex / negative regulation of t-circle formation / positive regulation of platelet formation / DNA end binding / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity ...Ku70:Ku80 complex / negative regulation of t-circle formation / positive regulation of platelet formation / DNA end binding / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / cellular response to X-ray / immunoglobulin V(D)J recombination / nonhomologous end joining complex / DNA ligation / nuclear telomere cap complex / regulation of smooth muscle cell proliferation / double-strand break repair via alternative nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / double-strand break repair via classical nonhomologous end joining / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / recombinational repair / regulation of telomere maintenance / U3 snoRNA binding / regulation of hematopoietic stem cell differentiation / positive regulation of neurogenesis / protein localization to chromosome, telomeric region / cellular response to fatty acid / hematopoietic stem cell proliferation / cellular hyperosmotic salinity response / maturation of 5.8S rRNA / T cell lineage commitment / negative regulation of cGAS/STING signaling pathway / telomeric DNA binding / positive regulation of catalytic activity / B cell lineage commitment / 2-LTR circle formation / positive regulation of double-strand break repair via nonhomologous end joining / site of DNA damage / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / enzyme activator activity / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / ectopic germ cell programmed cell death / ATP-dependent activity, acting on DNA / somitogenesis / positive regulation of telomerase activity / mitotic G1 DNA damage checkpoint signaling / positive regulation of telomere maintenance via telomerase / DNA helicase activity / telomere maintenance / activation of innate immune response / cellular response to leukemia inhibitory factor / neurogenesis / small-subunit processome / cyclin binding / positive regulation of translation / negative regulation of protein phosphorylation / positive regulation of erythrocyte differentiation / protein-DNA complex / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / brain development / peptidyl-threonine phosphorylation / protein destabilization / protein modification process / cellular response to gamma radiation / regulation of circadian rhythm / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / rhythmic process / double-strand break repair / intrinsic apoptotic signaling pathway in response to DNA damage / E3 ubiquitin ligases ubiquitinate target proteins / T cell differentiation in thymus / heart development / double-stranded DNA binding / scaffold protein binding / peptidyl-serine phosphorylation / secretory granule lumen / DNA recombination / ficolin-1-rich granule lumen / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / chromosome, telomeric region / damaged DNA binding / transcription cis-regulatory region binding / non-specific serine/threonine protein kinase / protein kinase activity / response to xenobiotic stimulus / ribonucleoprotein complex / positive regulation of apoptotic process / protein domain specific binding / protein phosphorylation
Similarity search - Function
Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain ...Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain / Ku70/Ku80 C-terminal arm / Ku70/Ku80 N-terminal alpha/beta domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / DNA-dependent protein kinase catalytic subunit, CC3 / SPOC-like, C-terminal domain superfamily / 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 / SAP domain superfamily / SAP domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / PIK-related kinase, FAT / FAT domain / FATC 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 / von Willebrand factor (vWF) type A domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic 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
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

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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
Water0
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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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 FIELDBright-field microscopy / 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
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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