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- PDB-7nfc: Cryo-EM structure of NHEJ super-complex (dimer) -

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Entry
Database: PDB / ID: 7nfc
TitleCryo-EM structure of NHEJ super-complex (dimer)
Components
  • (DNA (27-MER)) x 2
  • (X-ray repair cross-complementing protein ...) x 2
  • DNA (28-MER)
  • DNA ligase 4
  • DNA repair protein XRCC4
  • DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs
  • Non-homologous end-joining factor 1
KeywordsDNA BINDING PROTEIN / NHEJ / DNA-PKcs / Ku70/80 / XLF / XRCC4 / DNA-LigaseIV
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 / double-strand break repair via alternative nonhomologous end joining / regulation of epithelial cell proliferation / isotype switching / double-strand break repair via classical nonhomologous end joining / protein localization to site of double-strand break / nuclear telomere cap complex / 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 / U3 snoRNA binding / DNA biosynthetic process / protein localization to chromosome, telomeric region / T cell lineage commitment / maturation of 5.8S rRNA / cellular response to lithium ion / cellular hyperosmotic salinity response / positive regulation of double-strand break repair via nonhomologous end joining / negative regulation of cGAS/STING signaling pathway / B cell lineage commitment / response to ionizing radiation / 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 / stem cell proliferation / positive regulation of translation / cellular response to leukemia inhibitory factor / cellular response to ionizing radiation / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / small-subunit processome / enzyme activator activity / protein-DNA complex / cellular response to gamma radiation / peptidyl-serine phosphorylation / brain development / regulation of circadian rhythm / base-excision repair / 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 / 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 / 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: 4.14 Å
AuthorsChaplin, A.K. / Hardwick, S.W. / Kefala Stavridi, A. / Chirgadze, D.Y. / Blundell, T.L.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z; 2016 United Kingdom
CitationJournal: Mol Cell / Year: 2021
Title: Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.
Authors: Amanda K Chaplin / Steven W Hardwick / Antonia Kefala Stavridi / Christopher J Buehl / Noah J Goff / Virginie Ropars / Shikang Liang / Taiana Maia De Oliveira / Dimitri Y Chirgadze / ...Authors: Amanda K Chaplin / Steven W Hardwick / Antonia Kefala Stavridi / Christopher J Buehl / Noah J Goff / Virginie Ropars / Shikang Liang / Taiana Maia De Oliveira / Dimitri Y Chirgadze / Katheryn Meek / Jean-Baptiste Charbonnier / Tom L Blundell /
Abstract: Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or ...Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.
History
DepositionFeb 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 22, 2021Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jul 10, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / em_admin
Item: _citation.page_last / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs
B: X-ray repair cross-complementing protein 6
C: X-ray repair cross-complementing protein 5
F: DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs
G: X-ray repair cross-complementing protein 6
H: X-ray repair cross-complementing protein 5
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
D: DNA (27-MER)
E: DNA (28-MER)
I: DNA (28-MER)
J: DNA (27-MER)


Theoretical massNumber of molelcules
Total (without water)1,710,53618
Polymers1,710,53618
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area87710 Å2
ΔGint-473 kcal/mol
Surface area536560 Å2
MethodPISA

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Components

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Protein , 4 types, 10 molecules AFKLNOMPQR

#1: Protein DNA-dependent protein kinase catalytic subunit,DNA-dependent protein kinase catalytic subunit,DNA-PKcs / DNA-PK catalytic subunit / DNA-PKcs / DNPK1 / p460


Mass: 471375.406 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
#4: 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
#5: 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)
#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 BL21(DE3) (bacteria) / References: UniProt: Q9H9Q4

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

#2: 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: 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
#3: 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: 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 , 3 types, 4 molecules DEIJ

#7: DNA chain DNA (27-MER)


Mass: 8335.403 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#8: DNA chain DNA (28-MER)


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


Mass: 8350.414 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

Component
IDNameTypeEntity IDParent-IDSource
1NHEJ super-complex (dimer)ORGANELLE OR CELLULAR COMPONENTall0MULTIPLE SOURCES
2DNA-dependent protein kinase catalytic subunitORGANELLE OR CELLULAR COMPONENT#11NATURAL
3X-ray repair cross-complementing proteins 5 and 6ORGANELLE OR CELLULAR COMPONENT#2-#31RECOMBINANT
4Repair protein and ligaseORGANELLE OR CELLULAR COMPONENT#4-#61RECOMBINANT
5DNAORGANELLE OR CELLULAR COMPONENT#7-#91RECOMBINANT
Molecular weightValue: 1.6 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
45Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Insect cell expression vector pTIE1 (others)266783
24Escherichia coli BL21(DE3) (bacteria)469008
35synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.3 sec. / Electron dose: 46.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19rc7_4070refinement
PHENIX1.19rc7_4070refinement
EM software
IDNameCategory
2EPUimage acquisition
4WarpCTF correction
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: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23421 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 294.33 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00391107
ELECTRON MICROSCOPYf_angle_d0.6422123601
ELECTRON MICROSCOPYf_chiral_restr0.039114044
ELECTRON MICROSCOPYf_plane_restr0.003715376
ELECTRON MICROSCOPYf_dihedral_angle_d15.580434183

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