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- PDB-8ezb: NHEJ Long-range complex with ATP -

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Basic information

Entry
Database: PDB / ID: 8ezb
TitleNHEJ Long-range complex with ATP
Components
  • (X-ray repair cross-complementing protein ...) x 2
  • DNA (30-MER)
  • DNA (31-MER)
  • DNA ligase 4
  • DNA repair protein XRCC4
  • DNA-dependent protein kinase catalytic subunit
  • Non-homologous end-joining factor 1
  • Protein PAXX
KeywordsDNA BINDING PROTEIN/DNA / Complex / Kinase / NHEJ / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / DNA ligase IV complex / positive regulation of platelet formation / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / DNA ligase (ATP) / negative regulation of t-circle formation ...FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / DNA ligase IV complex / positive regulation of platelet formation / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / DNA ligase (ATP) / 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 ligase (ATP) activity / 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 / nucleotide-excision repair, DNA gap filling / immature B cell differentiation / single strand break repair / regulation of smooth muscle cell proliferation / cellular response to X-ray / V(D)J recombination / 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 / isotype switching / Cytosolic sensors of pathogen-associated DNA / protein localization to site of double-strand break / telomere capping / IRF3-mediated induction of type I IFN / regulation of hematopoietic stem cell differentiation / positive regulation of neurogenesis / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / U3 snoRNA binding / DNA biosynthetic process / maturation of 5.8S rRNA / T cell lineage commitment / cellular response to lithium ion / cellular hyperosmotic salinity response / negative regulation of cGAS/STING signaling pathway / positive regulation of double-strand break repair via nonhomologous end joining / response to ionizing radiation / 2-LTR circle formation / B cell lineage commitment / hematopoietic stem cell proliferation / telomeric DNA binding / ligase activity / 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 / T cell differentiation / 5'-deoxyribose-5-phosphate lyase activity / somatic stem cell population maintenance / hematopoietic stem cell differentiation / response to X-ray / chromosome organization / ATP-dependent activity, acting on DNA / ectopic germ cell programmed cell death / somitogenesis / telomere maintenance via telomerase / SUMOylation of DNA damage response and repair proteins / condensed chromosome / DNA polymerase binding / mitotic G1 DNA damage checkpoint signaling / neurogenesis / activation of innate immune response / telomere maintenance / DNA helicase activity / cyclin binding / positive regulation of erythrocyte differentiation / cellular response to leukemia inhibitory factor / positive regulation of translation / B cell differentiation / central nervous system development / stem cell proliferation / response to gamma radiation / cellular response to ionizing radiation / small-subunit processome / Nonhomologous End-Joining (NHEJ) / enzyme activator activity / cellular response to gamma radiation / protein-DNA complex / base-excision repair / regulation of circadian rhythm / brain development / peptidyl-serine phosphorylation / protein destabilization / protein modification process / double-strand break repair via nonhomologous end joining
Similarity search - Function
Protein PAXX / : / PAXX, PAralog of XRCC4 and XLF, also called C9orf142 / XLF, N-terminal / : / : / XLF N-terminal domain / XLF protein coiled-coil region / DNA ligase IV domain / DNA ligase IV ...Protein PAXX / : / PAXX, PAralog of XRCC4 and XLF, also called C9orf142 / XLF, N-terminal / : / : / XLF N-terminal domain / XLF protein coiled-coil region / DNA ligase IV domain / DNA ligase IV / DNA ligase 4 / DNA Ligase 4, adenylation domain / XRCC4, N-terminal domain superfamily / DNA repair protein XRCC4 / : / : / : / XRCC4 N-terminal domain / XRCC4 coiled-coil / XRCC4 C-terminal region / XRCC4-like, N-terminal domain superfamily / 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 / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / 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 / ATP-dependent DNA ligase AMP-binding site. / ATP-dependent DNA ligase signature 2. / DNA ligase, ATP-dependent, C-terminal / ATP dependent DNA ligase C terminal region / DNA ligase, ATP-dependent, conserved site / ATP-dependent DNA ligase family profile. / DNA ligase, ATP-dependent, central / ATP dependent DNA ligase domain / SAP domain superfamily / DNA repair protein XRCC4-like, C-terminal / 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. / BRCA1 C Terminus (BRCT) domain / Phosphatidylinositol 3- and 4-kinases signature 1. / breast cancer carboxy-terminal domain / 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 / BRCT domain profile. / BRCT domain / BRCT domain superfamily / von Willebrand factor A-like domain superfamily / Armadillo-like helical / Armadillo-type fold / Nucleic acid-binding, OB-fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5 / DNA ligase 4 / DNA-dependent protein kinase catalytic subunit / DNA repair protein XRCC4 / Protein PAXX / Non-homologous end-joining factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsChen, S. / He, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 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

Downloads & links

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Assembly

Deposited unit
A: X-ray repair cross-complementing protein 6
B: X-ray repair cross-complementing protein 5
C: DNA-dependent protein kinase catalytic subunit
D: DNA (31-MER)
E: DNA (30-MER)
F: DNA repair protein XRCC4
G: DNA repair protein XRCC4
H: Non-homologous end-joining factor 1
I: Non-homologous end-joining factor 1
J: X-ray repair cross-complementing protein 6
K: X-ray repair cross-complementing protein 5
L: DNA-dependent protein kinase catalytic subunit
M: DNA (31-MER)
N: DNA (30-MER)
O: DNA repair protein XRCC4
P: DNA repair protein XRCC4
S: Protein PAXX
T: Protein PAXX
X: DNA ligase 4
Y: DNA ligase 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,755,09124
Polymers1,754,02820
Non-polymers1,0634
Water00
1


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

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Components

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X-ray repair cross-complementing protein ... , 2 types, 4 molecules AJBK

#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: 69945.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1 / Production host: Spodoptera frugiperda (fall armyworm)
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: 82812.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Protein , 5 types, 12 molecules CLFGOPHISTXY

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


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, non-specific serine/threonine protein kinase
#6: Protein
DNA repair protein XRCC4 / X-ray repair cross-complementing protein 4


Mass: 38337.703 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13426
#7: Protein Non-homologous end-joining factor 1 / Protein cernunnos / XRCC4-like factor


Mass: 33372.234 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NHEJ1, XLF / Production host: Escherichia coli (E. coli) / References: UniProt: Q9H9Q4
#8: Protein Protein PAXX / Paralog of XRCC4 and XLF / XRCC4-like small protein


Mass: 21663.498 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PAXX, C9orf142, XLS / Production host: Escherichia coli (E. coli) / References: UniProt: Q9BUH6
#9: Protein DNA ligase 4 / DNA ligase IV / Polydeoxyribonucleotide synthase [ATP] 4


Mass: 104124.953 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LIG4 / Production host: Escherichia coli (E. coli) / References: UniProt: P49917, DNA ligase (ATP)

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DNA chain , 2 types, 4 molecules DMEN

#4: DNA chain DNA (31-MER)


Mass: 9510.159 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain DNA (30-MER)


Mass: 9236.976 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 4 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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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: NHEJ Long-range complex with ATP / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 1.75 MDa / Experimental value: NO
Buffer solutionpH: 7.9
SpecimenConc.: 2 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Calibrated magnification: 60000 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: 2 / Num. of real images: 30448
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.8particle selection
2cryoSPARC2image acquisition
4RELION3.1.3CTF correction
7ISOLDE1.3model fitting
9ISOLDE1.3model refinement
10RELION3.1.3initial Euler assignment
11RELION3.1.3final Euler assignment
12RELION3.1.3classification
13RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3785786
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 495819 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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