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- PDB-8ez9: Dimeric complex of DNA-PKcs -

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Basic information

Entry
Database: PDB / ID: 8ez9
TitleDimeric complex of DNA-PKcs
Components
  • DNA-dependent protein kinase catalytic subunit
  • unknown region of DNA-PKcs
KeywordsDNA BINDING PROTEIN / Dimer / Kinase / Complex / NHEJ
Function / homology
Function and homology information


positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immature B cell differentiation / nonhomologous end joining complex / immunoglobulin V(D)J recombination ...positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immature B cell differentiation / nonhomologous end joining complex / immunoglobulin V(D)J recombination / regulation of smooth muscle cell proliferation / Cytosolic sensors of pathogen-associated DNA / double-strand break repair via alternative nonhomologous end joining / IRF3-mediated induction of type I IFN / telomere capping / regulation of epithelial cell proliferation / regulation of hematopoietic stem cell differentiation / U3 snoRNA binding / T cell lineage commitment / maturation of 5.8S rRNA / negative regulation of cGAS/STING signaling pathway / positive regulation of double-strand break repair via nonhomologous end joining / B cell lineage commitment / ectopic germ cell programmed cell death / somitogenesis / negative regulation of protein phosphorylation / mitotic G1 DNA damage checkpoint signaling / telomere maintenance / activation of innate immune response / positive regulation of erythrocyte differentiation / positive regulation of translation / response to gamma radiation / peptidyl-threonine phosphorylation / small-subunit processome / Nonhomologous End-Joining (NHEJ) / protein-DNA complex / regulation of circadian rhythm / protein modification process / protein destabilization / brain development / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / intrinsic apoptotic signaling pathway in response to DNA damage / rhythmic process / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / heart development / T cell differentiation in thymus / peptidyl-serine phosphorylation / double-stranded DNA binding / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / eukaryotic translation initiation factor 2alpha kinase activity / chromosome, telomeric region / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H3T6 kinase activity / histone H2AS1 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / non-specific serine/threonine protein kinase / protein kinase activity / protein phosphorylation / positive regulation of apoptotic process / protein domain specific binding / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / negative regulation of apoptotic process / nucleolus / chromatin / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / RNA binding / nucleoplasm / ATP binding / nucleus / membrane / cytosol
Similarity search - Function
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 ...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 / : / 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 / Armadillo-like helical / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
DNA-dependent protein kinase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.67 Å
AuthorsChen, S. / He, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM135651 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.
Authors: Siyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
Abstract: DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). ...DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen ). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.
History
DepositionOct 31, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: unknown region of DNA-PKcs
L: DNA-dependent protein kinase catalytic subunit
C: DNA-dependent protein kinase catalytic subunit
Q: unknown region of DNA-PKcs


Theoretical massNumber of molelcules
Total (without water)942,7874
Polymers942,7874
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide unknown region of DNA-PKcs


Mass: 1720.111 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA / References: non-specific serine/threonine protein kinase
#2: Protein DNA-dependent protein kinase catalytic subunit


Mass: 469673.219 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA / References: UniProt: P78527

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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: dimer of DNA-PKcs / Type: COMPLEX / Entity ID: #2, #1 / Source: NATURAL
Molecular weightValue: 0.94 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 302 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 4000 nm / Cs: 2 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 4 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13132
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.4/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDNameVersionCategory
1Gautomatch0.8particle selection
2cryoSPARC2image acquisition
4RELION3.1.3CTF correction
7ISOLDE1.4model fitting
9RELION3.1.3initial Euler assignment
10RELION3.1.3final Euler assignment
11RELION3.1.3classification
12RELION3.1.33D reconstruction
13ISOLDE1.4model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1587734
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 5.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64775 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00429994
ELECTRON MICROSCOPYf_angle_d0.88340551
ELECTRON MICROSCOPYf_dihedral_angle_d7.7023966
ELECTRON MICROSCOPYf_chiral_restr0.0484604
ELECTRON MICROSCOPYf_plane_restr0.0065161

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