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- PDB-6zha: Cryo-EM structure of DNA-PK monomer -

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Basic information

Entry
Database: PDB / ID: 6zha
TitleCryo-EM structure of DNA-PK monomer
Components
  • (DNA) x 2
  • DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs
  • X-ray repair cross-complementing protein 5
  • X-ray repair cross-complementing protein 6
KeywordsDNA BINDING PROTEIN / Kinase / DNA-PKcs / NHEJ / DNA-repair / DNA-PK / Ku70/80
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 / small-subunit processome / neurogenesis / cyclin binding / negative regulation of protein phosphorylation / positive regulation of erythrocyte differentiation / positive regulation of translation / 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 modification process / protein destabilization / cellular response to gamma radiation / regulation of circadian rhythm / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / rhythmic process / intrinsic apoptotic signaling pathway in response to DNA damage / double-strand break repair / 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 / damaged DNA binding / chromosome, telomeric region / 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 / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / von Willebrand factor (vWF) type A domain / 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: 3.91 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 21, 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

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Structure visualization

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  • Deposited structure unit
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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-PKcs
B: X-ray repair cross-complementing protein 6
C: X-ray repair cross-complementing protein 5
D: DNA
E: DNA


Theoretical massNumber of molelcules
Total (without water)639,8895
Polymers639,8895
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25370 Å2
ΔGint-151 kcal/mol
Surface area212550 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-PKcs / DNA-PKcs / DNPK1 / p460


Mass: 472056.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA
References: UniProt: P78527, non-specific serine/threonine protein kinase
#2: 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: 69945.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1
Production host: Insect cell expression vector pTIE1 (others)
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
#3: 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: 82812.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#4: DNA chain DNA /


Mass: 7672.036 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain DNA /


Mass: 7402.821 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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

Component
IDNameTypeEntity IDParent-IDSource
1DNA-PK monomerCOMPLEXall0MULTIPLE SOURCES
2DNA-dependent protein kinase catalytic subunitCOMPLEX#11NATURAL
3X-ray repair cross-complementing protein 5+6COMPLEX#2-#31RECOMBINANT
4DNACOMPLEX#4-#51RECOMBINANT
Molecular weightValue: 0.64 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Insect cell expression vector pTIE1 (others)266783
24synthetic construct (others)32630
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 FIELDBright-field microscopy
Image recordingElectron dose: 52.97 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1Warpparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 350668
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59967 / Symmetry type: POINT

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