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- PDB-7zyg: CryoEM structure of Ku heterodimer bound to DNA, PAXX and XLF -

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Basic information

Entry
Database: PDB / ID: 7zyg
TitleCryoEM structure of Ku heterodimer bound to DNA, PAXX and XLF
Components
  • (DNA) x 2
  • (X-ray repair cross-complementing protein ...) x 2
  • Non-homologous end-joining factor 1
  • Protein PAXX
KeywordsDNA BINDING PROTEIN / NHEJ / Ku70 / Ku80 / DNA damage / XLF
Function / homology
Function and homology information


positive regulation of ligase activity / DNA ligase IV complex / Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination ...positive regulation of ligase activity / DNA ligase IV complex / Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / cellular response to X-ray / nuclear telomere cap complex / double-strand break repair via classical nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / positive regulation of neurogenesis / recombinational repair / regulation of telomere maintenance / U3 snoRNA binding / protein localization to chromosome, telomeric region / cellular hyperosmotic salinity response / response to ionizing radiation / 2-LTR circle formation / telomeric DNA binding / hematopoietic stem cell proliferation / positive regulation of protein kinase activity / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / site of DNA damage / T cell differentiation / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / ATP-dependent activity, acting on DNA / telomere maintenance via telomerase / DNA polymerase binding / neurogenesis / telomere maintenance / activation of innate immune response / DNA helicase activity / cyclin binding / cellular response to leukemia inhibitory factor / central nervous system development / B cell differentiation / small-subunit processome / enzyme activator activity / Nonhomologous End-Joining (NHEJ) / cellular response to gamma radiation / protein-DNA complex / double-strand break repair via nonhomologous end joining / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / fibrillar center / double-strand break repair / site of double-strand break / double-stranded DNA binding / scaffold protein binding / secretory granule lumen / DNA recombination / transcription regulator complex / molecular adaptor activity / ficolin-1-rich granule lumen / damaged DNA binding / chromosome, telomeric region / transcription cis-regulatory region binding / ribonucleoprotein complex / innate immune response / negative regulation of DNA-templated transcription / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / positive regulation of DNA-templated transcription / protein-containing complex binding / nucleolus / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / protein-containing complex / ATP hydrolysis activity / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Ku70; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / Protein PAXX / : / PAXX, PAralog of XRCC4 and XLF, also called C9orf142 / XLF, N-terminal / : / : / XLF N-terminal domain / XLF protein coiled-coil region ...Ku70; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / Protein PAXX / : / PAXX, PAralog of XRCC4 and XLF, also called C9orf142 / XLF, N-terminal / : / : / XLF N-terminal domain / XLF protein coiled-coil 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 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 / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / von Willebrand factor, type A domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / DNA / DNA (> 10) / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5 / Protein PAXX / Non-homologous end-joining factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsHardwick, S.W. / Kefala-Stavridi, A. / Chirgadze, D.Y. / Blundell, T.L. / Chaplin, A.K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200814/Z/16/Z; 2016 United Kingdom
CitationJournal: Sci Adv / Year: 2023
Title: PAXX binding to the NHEJ machinery explains functional redundancy with XLF.
Authors: Murielle Seif-El-Dahan / Antonia Kefala-Stavridi / Philippe Frit / Steven W Hardwick / Dima Y Chirgadze / Taiana Maia De Oliviera / Jessica Andreani / Sébastien Britton / Nadia Barboule / ...Authors: Murielle Seif-El-Dahan / Antonia Kefala-Stavridi / Philippe Frit / Steven W Hardwick / Dima Y Chirgadze / Taiana Maia De Oliviera / Jessica Andreani / Sébastien Britton / Nadia Barboule / Madeleine Bossaert / Arun Prasad Pandurangan / Katheryn Meek / Tom L Blundell / Virginie Ropars / Patrick Calsou / Jean-Baptiste Charbonnier / Amanda K Chaplin /
Abstract: Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX ...Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells.
History
DepositionMay 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 2.0Jun 14, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / em_entity_assembly ...atom_site / em_entity_assembly / em_entity_assembly_recombinant / entity / entity_poly / entity_poly_seq / entity_src_gen / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_entity_nonpoly / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly_gen / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_polymer_linkage / struct_asym / struct_conn / struct_ref_seq
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.group_PDB / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site.type_symbol / _em_entity_assembly.entity_id_list / _em_entity_assembly.source / _em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.organism / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_struct_assembly_gen.asym_id_list / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end
Revision 2.1Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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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: Protein PAXX
F: Non-homologous end-joining factor 1
D: DNA
E: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)217,0657
Polymers216,9706
Non-polymers951
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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X-ray repair cross-complementing protein ... , 2 types, 2 molecules AB

#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.: 1
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.: 1
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 , 2 types, 2 molecules CF

#3: Protein Protein PAXX / Paralog of XRCC4 and XLF / XRCC4-like small protein


Mass: 21663.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PAXX, C9orf142, XLS / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9BUH6
#4: Protein Non-homologous end-joining factor 1 / Protein cernunnos / XRCC4-like factor


Mass: 33372.234 Da / Num. of mol.: 1
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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DNA chain , 2 types, 2 molecules DE

#5: DNA chain DNA


Mass: 4503.948 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA


Mass: 4673.059 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4

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Details

Has ligand of interestN

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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: Ku70/80 hetero dimer bound to DNA, PAXX and XLF / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#4, #6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.16 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 2 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 / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.37 sec. / Electron dose: 52.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13024

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
7cryoSPARCmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1055761
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173112 / Symmetry type: POINT
Atomic model buildingPDB-ID: 1JEY

1jey


Accession code: 1JEY / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 162.51 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00199154
ELECTRON MICROSCOPYf_angle_d0.432812473
ELECTRON MICROSCOPYf_chiral_restr0.03921399
ELECTRON MICROSCOPYf_plane_restr0.00261502
ELECTRON MICROSCOPYf_dihedral_angle_d16.37041405

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