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Entry
Database: PDB / ID: 8bot
TitleCryo-EM structure of NHEJ supercomplex(trimer)
Components
  • (DNA (24-MER)) x 2
  • (X-ray repair cross-complementing protein ...) x 2
  • DNA (27-MER)
  • DNA (28-MER)
  • DNA ligase 4
  • DNA repair protein XRCC4
  • DNA-dependent protein kinase catalytic subunit
  • Non-homologous end-joining factor 1
KeywordsDNA BINDING PROTEIN / NHEJ / DNA-PK / DNA-PKcs / Ku70 / Ku80 / XLF / DNA repair
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 / DN2 thymocyte differentiation / DNA ligase (ATP) / Ku70:Ku80 complex / T cell receptor V(D)J recombination ...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 / DN2 thymocyte differentiation / DNA ligase (ATP) / Ku70:Ku80 complex / T cell receptor V(D)J recombination / negative regulation of t-circle formation / pro-B cell differentiation / DNA end binding / DNA ligase (ATP) activity / DNA-dependent protein kinase activity / small-subunit processome assembly / positive regulation of lymphocyte differentiation / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination / nonhomologous end joining complex / nucleotide-excision repair, DNA gap filling / single strand break repair / immature B cell differentiation / V(D)J recombination / regulation of smooth muscle cell proliferation / cellular response to X-ray / regulation of epithelial cell proliferation / double-strand break repair via alternative nonhomologous end joining / isotype switching / double-strand break repair via classical nonhomologous end joining / nuclear telomere cap complex / protein localization to site of double-strand break / Cytosolic sensors of pathogen-associated DNA / telomere capping / IRF3-mediated induction of type I IFN / regulation of hematopoietic stem cell differentiation / recombinational repair / positive regulation of neurogenesis / regulation of telomere maintenance / DNA biosynthetic process / U3 snoRNA binding / protein localization to chromosome, telomeric region / 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 / B cell lineage commitment / 2-LTR circle formation / hematopoietic stem cell proliferation / ligase activity / telomeric DNA binding / 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 / somatic stem cell population maintenance / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / response to X-ray / chromosome organization / ATP-dependent activity, acting on DNA / somitogenesis / ectopic germ cell programmed cell death / 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 / DNA helicase activity / telomere maintenance / positive regulation of erythrocyte differentiation / B cell differentiation / cyclin binding / central nervous system development / positive regulation of translation / stem cell proliferation / cellular response to leukemia inhibitory factor / response to gamma radiation / cellular response to ionizing radiation / Nonhomologous End-Joining (NHEJ) / small-subunit processome / enzyme activator activity / cellular response to gamma radiation / protein-DNA complex / regulation of circadian rhythm / peptidyl-serine phosphorylation / base-excision repair / brain development / protein destabilization / protein modification process / double-strand break repair via nonhomologous end joining
Similarity search - Function
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 ...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 / 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, bridge and pillars domain superfamily / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / : / DNA ligase N terminus / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / ATP-dependent DNA ligase AMP-binding site. / ATP-dependent DNA ligase signature 2. / DNA ligase, ATP-dependent, C-terminal / Ku80 / ATP dependent DNA ligase C terminal region / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 N-terminal alpha/beta domain / DNA ligase, ATP-dependent, conserved site / ATP-dependent DNA ligase family profile. / 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 / 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 / breast cancer carboxy-terminal domain / 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 / BRCT domain profile. / von Willebrand factor, type A / 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
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 / Non-homologous end-joining factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.76 Å
AuthorsHardwick, S.W. / Chaplin, A.K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Structure / Year: 2023
Title: Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ.
Authors: Steven W Hardwick / Antonia Kefala Stavridi / Dimitri Y Chirgadze / Taiana Maia De Oliveira / Jean-Baptiste Charbonnier / Virginie Ropars / Katheryn Meek / Tom L Blundell / Amanda K Chaplin /
Abstract: The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ...The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.
History
DepositionNov 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
K: DNA repair protein XRCC4
L: DNA repair protein XRCC4
M: DNA ligase 4
N: DNA repair protein XRCC4
O: DNA repair protein XRCC4
P: DNA ligase 4
Q: Non-homologous end-joining factor 1
R: Non-homologous end-joining factor 1
F: DNA-dependent protein kinase catalytic subunit
G: X-ray repair cross-complementing protein 6
H: X-ray repair cross-complementing protein 5
I: DNA (28-MER)
J: DNA (27-MER)
A: DNA-dependent protein kinase catalytic subunit
B: X-ray repair cross-complementing protein 6
C: X-ray repair cross-complementing protein 5
D: DNA (24-MER)
E: DNA (24-MER)
S: DNA-dependent protein kinase catalytic subunit
T: X-ray repair cross-complementing protein 6
U: X-ray repair cross-complementing protein 5
V: DNA (28-MER)
W: DNA (27-MER)
X: Non-homologous end-joining factor 1
Y: Non-homologous end-joining factor 1


Theoretical massNumber of molelcules
Total (without water)2,411,09225
Polymers2,411,09225
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 13 molecules KLNOMPQRXYFAS

#1: 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 BL21(DE3) (bacteria) / References: UniProt: Q13426
#2: 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 BL21(DE3) (bacteria) / References: UniProt: P49917, DNA ligase (ATP)
#3: Protein
Non-homologous end-joining factor 1 / Protein cernunnos / XRCC4-like factor


Mass: 33372.234 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NHEJ1, XLF / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9H9Q4
#4: Protein DNA-dependent protein kinase catalytic subunit / DNA-PK catalytic subunit / DNA-PKcs / DNPK1 / p460


Mass: 469673.219 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P78527, non-specific serine/threonine protein kinase

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X-ray repair cross-complementing protein ... , 2 types, 6 molecules GBTHCU

#5: 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.: 3
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
#6: 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.: 3
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

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DNA chain , 4 types, 6 molecules IVJWDE

#7: DNA chain DNA (28-MER)


Mass: 8619.629 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#8: DNA chain DNA (27-MER)


Mass: 8350.414 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#9: DNA chain DNA (24-MER)


Mass: 7367.843 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#10: DNA chain DNA (24-MER)


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

ComponentName: NHEJ supercomplex trimer / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Trimeric complex of DNA-PK, DNA ligase 4, XRCC4, XLF
Entity ID: all / Source: NATURAL
Molecular weightValue: 2.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 46.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 749185
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9114 / Symmetry type: POINT
Atomic model buildingB value: 639
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 636.65 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039132129
ELECTRON MICROSCOPYf_angle_d0.8218179384
ELECTRON MICROSCOPYf_chiral_restr0.046920511
ELECTRON MICROSCOPYf_plane_restr0.004922360
ELECTRON MICROSCOPYf_dihedral_angle_d15.916749261

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