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- PDB-7lsy: NHEJ Short-range synaptic complex -

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Basic information

Entry
Database: PDB / ID: 7lsy
TitleNHEJ Short-range synaptic complex
Components
  • (DNA (26-MER)) x 2
  • (X-ray repair cross-complementing protein ...) x 2
  • DNA (5'-D(P*CP*AP*AP*TP*GP*AP*AP*AP*CP*GP*GP*AP*AP*CP*AP*GP*TP*CP*AP*G)-3')
  • DNA (5'-D(P*GP*TP*TP*CP*TP*TP*AP*GP*TP*AP*TP*AP*TP*A)-3')
  • DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')
  • DNA ligase 4
  • DNA repair protein XRCC4
  • Non-homologous end-joining factor 1
KeywordsDNA BINDING PROTEIN/DNA / NHEJ / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA ligation involved in DNA recombination / T cell receptor V(D)J recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / pro-B cell differentiation / DNA ligase IV complex / positive regulation of lymphocyte differentiation / DNA ligation involved in DNA repair / small-subunit processome assembly ...DNA ligation involved in DNA recombination / T cell receptor V(D)J recombination / FHA domain binding / positive regulation of chromosome organization / positive regulation of ligase activity / pro-B cell differentiation / DNA ligase IV complex / positive regulation of lymphocyte differentiation / DNA ligation involved in DNA repair / small-subunit processome assembly / DNA ligase activity / Ku70:Ku80 complex / DN2 thymocyte differentiation / DNA-dependent protein kinase complex / immunoglobulin V(D)J recombination / negative regulation of t-circle formation / DNA end binding / DNA ligase (ATP) / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / DNA ligase (ATP) activity / cellular response to X-ray / regulation of smooth muscle cell proliferation / single strand break repair / Cytosolic sensors of pathogen-associated DNA / nucleotide-excision repair, DNA gap filling / DNA ligation / V(D)J recombination / IRF3-mediated induction of type I IFN / nuclear telomere cap complex / double-strand break repair via classical nonhomologous end joining / isotype switching / protein localization to site of double-strand break / positive regulation of catalytic activity / U3 snoRNA binding / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / cellular response to fatty acid / positive regulation of neurogenesis / cellular hyperosmotic salinity response / hematopoietic stem cell proliferation / response to ionizing radiation / cellular response to lithium ion / DNA biosynthetic process / telomeric DNA binding / 2-LTR circle formation / ligase activity / : / site of DNA damage / somatic stem cell population maintenance / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / response to X-ray / T cell differentiation / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / chromosome organization / ATP-dependent activity, acting on DNA / SUMOylation of DNA damage response and repair proteins / DNA polymerase binding / condensed chromosome / activation of innate immune response / enzyme activator activity / positive regulation of telomere maintenance via telomerase / DNA helicase activity / telomere maintenance / cyclin binding / B cell differentiation / neurogenesis / protein-DNA complex / stem cell proliferation / response to gamma radiation / cellular response to leukemia inhibitory factor / central nervous system development / small-subunit processome / cellular response to ionizing radiation / Nonhomologous End-Joining (NHEJ) / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to gamma radiation / fibrillar center / double-strand break repair via nonhomologous end joining / establishment of integrated proviral latency / positive regulation of fibroblast proliferation / double-strand break repair / site of double-strand break / T cell differentiation in thymus / double-stranded DNA binding / fibroblast proliferation / scaffold protein binding / secretory granule lumen / DNA recombination / in utero embryonic development / neuron apoptotic process / negative regulation of neuron apoptotic process / transcription regulator complex / ficolin-1-rich granule lumen / cell population proliferation / damaged DNA binding / chromosome, telomeric region
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 / SPOC-like, C-terminal domain superfamily / 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. / SAP domain superfamily / DNA ligase, ATP-dependent, central / ATP dependent DNA ligase domain / DNA repair protein XRCC4-like, C-terminal / SAP domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal 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 / Nucleic acid-binding, OB-fold
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 repair protein XRCC4 / Non-homologous end-joining factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.4 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
American Cancer SocietyIRG-15-173-2 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P01CA092584 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 1.3Aug 16, 2023Group: Data collection / Database references / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.pdb_format_compatible

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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
D: DNA (26-MER)
E: DNA (5'-D(P*GP*TP*TP*CP*TP*TP*AP*GP*TP*AP*TP*AP*TP*A)-3')
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
M: DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')
N: DNA (26-MER)
O: DNA repair protein XRCC4
P: DNA repair protein XRCC4
V: DNA (5'-D(P*CP*AP*AP*TP*GP*AP*AP*AP*CP*GP*GP*AP*AP*CP*AP*GP*TP*CP*AP*G)-3')
X: DNA ligase 4
Y: DNA ligase 4


Theoretical massNumber of molelcules
Total (without water)762,35817
Polymers762,35817
Non-polymers00
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 / CTC75 / 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: 68872.875 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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DNA chain , 5 types, 5 molecules DEMNV

#3: DNA chain DNA (26-MER)


Mass: 7936.151 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: DNA chain DNA (5'-D(P*GP*TP*TP*CP*TP*TP*AP*GP*TP*AP*TP*AP*TP*A)-3')


Mass: 4284.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#7: DNA chain DNA (5'-D(P*TP*AP*TP*AP*TP*AP*CP*TP*AP*AP*GP*AP*AP*C)-3')


Mass: 4271.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#8: DNA chain DNA (26-MER)


Mass: 7964.145 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#9: DNA chain DNA (5'-D(P*CP*AP*AP*TP*GP*AP*AP*AP*CP*GP*GP*AP*AP*CP*AP*GP*TP*CP*AP*G)-3')


Mass: 6185.046 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein , 3 types, 8 molecules FGOPHIXY

#5: 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
#6: 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
#10: 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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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: Short-range synaptic complex of NHEJ / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.72 MDa / Experimental value: NO
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
250 mMPotassium chlorideKCl1
310 mMMagnesium chlorideMgCl21
42.5 %glycerol1
50.05 %NP-401
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 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

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: DARK FIELD / Nominal magnification: 30000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.0426 sec. / Electron dose: 46 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 32723
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
4GctfCTF correction
5CTFFINDCTF correction
8UCSF Chimeramodel fitting
9ISOLDEmodel fitting
10Cootmodel 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: 1766936
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175866 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient

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