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- PDB-8ouy: Human RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) complex, 3.4 A resolution -

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Basic information

Entry
Database: PDB / ID: 8ouy
TitleHuman RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) complex, 3.4 A resolution
Components
  • (DNA repair protein RAD51 homolog ...) x 3
  • DNA repair protein XRCC2
KeywordsDNA BINDING PROTEIN / Complex / ssDNA-binding / ATPase / RAD51
Function / homology
Function and homology information


meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion ...meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion / gamma-tubulin binding / reciprocal meiotic recombination / sister chromatid cohesion / regulation of fibroblast apoptotic process / centrosome cycle / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / positive regulation of neurogenesis / ATP-dependent DNA damage sensor activity / male meiosis I / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / somitogenesis / interstrand cross-link repair / four-way junction DNA binding / positive regulation of G2/M transition of mitotic cell cycle / telomere maintenance / neurogenesis / meiotic cell cycle / replication fork / response to gamma radiation / TP53 Regulates Transcription of DNA Repair Genes / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / multicellular organism growth / Meiotic recombination / cell junction / mitotic cell cycle / single-stranded DNA binding / Factors involved in megakaryocyte development and platelet production / spermatogenesis / double-stranded DNA binding / DNA recombination / in utero embryonic development / negative regulation of neuron apoptotic process / chromosome, telomeric region / regulation of cell cycle / intracellular membrane-bounded organelle / DNA repair / centrosome / positive regulation of cell population proliferation / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / DNA binding / nucleoplasm / ATP binding / nucleus / cytoplasm / cytosol
Similarity search - Function
DNA repair protein XRCC2 / DNA repair protein RAD51 homologue 2 / : / : / RAD51D, N-terminal domain / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. ...DNA repair protein XRCC2 / DNA repair protein RAD51 homologue 2 / : / : / RAD51D, N-terminal domain / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA repair protein RAD51 homolog 2 / DNA repair protein RAD51 homolog 3 / DNA repair protein XRCC2 / DNA repair protein RAD51 homolog 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGreenhough, L.A. / Liang, C.C. / West, S.C.
Funding support United Kingdom, European Union, 5items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W01355X/1 United Kingdom
Cancer Research UKCC2098 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2098 United Kingdom
Wellcome TrustCC2098 United Kingdom
European Research Council (ERC)ERC-ADG-666400European Union
CitationJournal: Nature / Year: 2023
Title: Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor.
Authors: Luke A Greenhough / Chih-Chao Liang / Ondrej Belan / Simone Kunzelmann / Sarah Maslen / Monica C Rodrigo-Brenni / Roopesh Anand / Mark Skehel / Simon J Boulton / Stephen C West /
Abstract: Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic ...Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers and the cancer-prone syndrome Fanconi anaemia. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.
History
DepositionApr 25, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 2, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair protein RAD51 homolog 2
B: DNA repair protein RAD51 homolog 3
C: DNA repair protein RAD51 homolog 4
D: DNA repair protein XRCC2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,13610
Polymers147,6214
Non-polymers1,5146
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14270 Å2
ΔGint-120 kcal/mol
Surface area39770 Å2

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Components

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DNA repair protein RAD51 homolog ... , 3 types, 3 molecules ABC

#1: Protein DNA repair protein RAD51 homolog 2 / R51H2 / RAD51 homolog B / Rad51B / RAD51-like protein 1


Mass: 38296.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51B, RAD51L1, REC2 / Plasmid: pBIG-BCDX2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O15315
#2: Protein DNA repair protein RAD51 homolog 3 / R51H3 / RAD51 homolog C / RAD51-like protein 2


Mass: 42244.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51C, RAD51L2 / Plasmid: pBIG-BCDX2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O43502
#3: Protein DNA repair protein RAD51 homolog 4 / R51H3 / RAD51 homolog D / RAD51-like protein 3 / TRAD


Mass: 35084.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51D, RAD51L3 / Plasmid: pBIG-BCDX2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75771

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Protein , 1 types, 1 molecules D

#4: Protein DNA repair protein XRCC2 / X-ray repair cross-complementing protein 2


Mass: 31995.525 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC2 / Plasmid: pBIG-BCDX2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O43543

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Non-polymers , 3 types, 6 molecules

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


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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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: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) / Type: COMPLEX
Details: Recombinant BCDX2 complexes purified from Sf9 insect cells, crosslinked with 0.005% glutaraldehyde (RT, 10 minutes) in the presence of 30 nt ssDNA, and vitrified in the presence of ADP.AlFx. ...Details: Recombinant BCDX2 complexes purified from Sf9 insect cells, crosslinked with 0.005% glutaraldehyde (RT, 10 minutes) in the presence of 30 nt ssDNA, and vitrified in the presence of ADP.AlFx. Map was enhanced using DeepEMhancer
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pBIG-BCDX2
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5 mM ADP.BeFx (0.5 mM ADP, 0.5 mM BeSO4, 10 mM NaF), 0.25 mM TCEP, 0.0005% Tween20
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHydroxyethylpiperazineHEPES1
2100 mMSodium chlorideNaCl1
32.5 mMMagnesium chlorideMgCl21
40.5 mMAdenosine diphoshpateADP1
50.5 mMBeryllium sulphateBeSO41
610 mMSodium fluorideNaF1
70.25 mMTris(2-carboxyethyl)phosphineTCEP1
80.0005 %Polysorbate 20Tween201
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 24449
Details: 12,555 movies were collected with zero tilt, and a further 11,894 movies with a 20 degree tilt

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
2EPU2.1image acquisition
4CTFFIND4CTF correction
7PHENIX1.2model fitting
9PHENIX1.2model refinement
10Coot0.9.8.7model refinement
11RELION4initial Euler assignment
12RELION4final Euler assignment
13RELION4classification
14RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4528940
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87572 / Algorithm: FOURIER SPACE / Details: Map was enhanced using DeepEMhancer / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: AlphaFold2 monomer predictions of RAD51B, RAD51C, RAD51D and XRCC2 were docked into the EM density using Dock and Rebuild in Phenix. ATP and Mg2+ were added, and the coordinates were ...Details: AlphaFold2 monomer predictions of RAD51B, RAD51C, RAD51D and XRCC2 were docked into the EM density using Dock and Rebuild in Phenix. ATP and Mg2+ were added, and the coordinates were iteratively real space refined using Phenix and manually modified in COOT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.12 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00237428
ELECTRON MICROSCOPYf_angle_d0.497310043
ELECTRON MICROSCOPYf_chiral_restr0.04231177
ELECTRON MICROSCOPYf_plane_restr0.00281257
ELECTRON MICROSCOPYf_dihedral_angle_d4.73751007

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