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- PDB-7lt3: NHEJ Long-range synaptic complex -

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Basic information

Entry
Database: PDB / ID: 7lt3
TitleNHEJ Long-range synaptic complex
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
  • Unknown peptide
KeywordsDNA BINDING PROTEIN/DNA / 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 / : / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / positive regulation of platelet formation ...: / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / DNA ligase IV complex / : / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / positive regulation of platelet formation / negative regulation of t-circle formation / DNA ligase (ATP) / DNA end binding / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA ligase (ATP) activity / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immature B cell differentiation / cellular response to X-ray / nonhomologous end joining complex / immunoglobulin V(D)J recombination / nucleotide-excision repair, DNA gap filling / single strand break repair / : / regulation of smooth muscle cell proliferation / V(D)J recombination / nuclear telomere cap complex / Cytosolic sensors of pathogen-associated DNA / double-strand break repair via alternative nonhomologous end joining / double-strand break repair via classical nonhomologous end joining / isotype switching / protein localization to site of double-strand break / IRF3-mediated induction of type I IFN / telomere capping / regulation of epithelial cell proliferation / recombinational repair / regulation of hematopoietic stem cell differentiation / regulation of telomere maintenance / U3 snoRNA binding / protein localization to chromosome, telomeric region / cellular hyperosmotic salinity response / cellular response to fatty acid / positive regulation of neurogenesis / T cell lineage commitment / maturation of 5.8S rRNA / cellular response to lithium ion / response to ionizing radiation / DNA biosynthetic process / telomeric DNA binding / negative regulation of cGAS/STING signaling pathway / positive regulation of double-strand break repair via nonhomologous end joining / B cell lineage commitment / hematopoietic stem cell proliferation / 2-LTR circle formation / ligase activity / site of DNA damage / somatic stem cell population maintenance / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / T cell differentiation / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / ATP-dependent activity, acting on DNA / positive regulation of protein kinase activity / response to X-ray / chromosome organization / ectopic germ cell programmed cell death / telomere maintenance via telomerase / somitogenesis / SUMOylation of DNA damage response and repair proteins / negative regulation of protein phosphorylation / DNA helicase activity / condensed chromosome / DNA polymerase binding / mitotic G1 DNA damage checkpoint signaling / telomere maintenance / cyclin binding / activation of innate immune response / enzyme activator activity / B cell differentiation / cellular response to leukemia inhibitory factor / positive regulation of erythrocyte differentiation / positive regulation of translation / stem cell proliferation / central nervous system development / neurogenesis / cellular response to ionizing radiation / response to gamma radiation / small-subunit processome / peptidyl-threonine phosphorylation / Nonhomologous End-Joining (NHEJ) / protein-DNA complex / cellular response to gamma radiation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / regulation of circadian rhythm / protein modification process / protein destabilization / brain development
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 / 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 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 beta-barrel domain / 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 ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / 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 / ATP-dependent DNA ligase signature 2. / ATP-dependent DNA ligase AMP-binding site. / 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 / SAP domain / DNA repair protein XRCC4-like, C-terminal / SAP motif profile. / 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 / 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'-DIPHOSPHATE / 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: 4.6 Å
AuthorsHe, Y. / Chen, S.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM135651 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM008382 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P01CA092584 United States
American Cancer SocietyIRG-15-173-21 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM047251 United States
CitationJournal: Nature / Year: 2021
Title: Structural basis of long-range to short-range synaptic transition in NHEJ.
Authors: Siyu Chen / Linda Lee / Tasmin Naila / Susan Fishbain / Annie Wang / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
Abstract: DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) ...DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.
History
DepositionFeb 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 26, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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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
Q: Unknown peptide
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
R: Unknown peptide
M: DNA (31-MER)
N: DNA (30-MER)
O: DNA repair protein XRCC4
P: DNA repair protein XRCC4
X: DNA ligase 4
Y: DNA ligase 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,714,99522
Polymers1,714,14120
Non-polymers8542
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Negative staining 2D class averages
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: unidentified baculovirus
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: unidentified baculovirus
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Protein , 4 types, 10 molecules CLFGOPHIXY

#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)
References: UniProt: P78527, non-specific serine/threonine protein kinase
#7: 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: unidentified baculovirus / References: UniProt: Q13426
#8: 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 BL21 (bacteria) / References: UniProt: Q9H9Q4
#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: unidentified baculovirus / References: UniProt: P49917, DNA ligase (ATP)

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DNA chain , 2 types, 4 molecules DMEN

#5: DNA chain DNA (31-MER)


Mass: 9510.159 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: 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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Protein/peptide / Non-polymers , 2 types, 4 molecules QR

#10: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Protein/peptide Unknown peptide


Mass: 1720.111 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: Maybe a part of PRKDC / Source: (natural) Homo sapiens (human)

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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: Long-range synaptic Complex of NHEJ / Type: COMPLEX / Details: DNAPKcs-Ku-XRCC4-LigIV-XLF / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 1.66 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
250 mMKCl1
310 mMMgCl21
42.5 %glycerol1
51 mMDTT1
60.05 %NP-401
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal magnification: 30000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 76.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 17114
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
2Appionimage acquisition
3RELION3.1image acquisition
5Gctf0.5CTF correction
6CTFFIND4.1CTF correction
9Cootmodel fitting
10ISOLDEmodel fitting
12EMANinitial Euler assignment
13cryoSPARCinitial Euler assignment
14RELION3.1final Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
17PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1119381
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329784 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16ERH

6erh

16ERH1PDBexperimental model
21JEY

1jey

11JEY2PDBexperimental model
35LUQ

5luq

15LUQ3PDBexperimental model
45Y3R

5y3r

15Y3R4PDBexperimental model
56ZHA

6zha

16ZHA5PDBexperimental model
62R9A

2r9a

12R9A6PDBexperimental model
73II6

3ii6

13II67PDBexperimental model

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