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Yorodumi- PDB-8ouz: Human RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) complex, 2.2 A resolution -
+Open data
-Basic information
Entry | Database: PDB / ID: 8ouz | ||||||||||||||||||
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Title | Human RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) complex, 2.2 A resolution | ||||||||||||||||||
Components |
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Keywords | DNA 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 / DNA strand invasion / Impaired BRCA2 binding to PALB2 ...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 / DNA strand invasion / Impaired BRCA2 binding to PALB2 / gamma-tubulin binding / reciprocal meiotic recombination / regulation of fibroblast apoptotic process / sister chromatid cohesion / 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 / positive regulation of G2/M transition of mitotic cell cycle / male meiosis I / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / neurogenesis / meiotic cell cycle / somitogenesis / interstrand cross-link repair / telomere maintenance / Meiotic recombination / replication fork / TP53 Regulates Transcription of DNA Repair Genes / response to gamma radiation / double-strand break repair via homologous recombination / multicellular organism growth / HDR through Homologous Recombination (HRR) / cell junction / Factors involved in megakaryocyte development and platelet production / double-stranded DNA binding / mitotic cell cycle / single-stranded DNA binding / chromosome, telomeric region / spermatogenesis / DNA recombination / in utero embryonic development / negative regulation of neuron apoptotic process / 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 | ||||||||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å | ||||||||||||||||||
Authors | Greenhough, L.A. / Liang, C.C. / West, S.C. | ||||||||||||||||||
Funding support | United Kingdom, European Union, 5items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ouz.cif.gz | 370.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8ouz.ent.gz | 294.7 KB | Display | PDB format |
PDBx/mmJSON format | 8ouz.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ouz_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8ouz_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8ouz_validation.xml.gz | 41.4 KB | Display | |
Data in CIF | 8ouz_validation.cif.gz | 62.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ou/8ouz ftp://data.pdbj.org/pub/pdb/validation_reports/ou/8ouz | HTTPS FTP |
-Related structure data
Related structure data | 17206MC 8ouyC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA repair protein RAD51 homolog ... , 3 types, 3 molecules ABC
#1: Protein | 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 |
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#2: Protein | 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 | 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 |
-Protein , 1 types, 1 molecules D
#4: Protein | Mass: 31921.381 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 , 4 types, 148 molecules
#5: Chemical | ChemComp-ADP / | ||||
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#6: Chemical | #7: Chemical | #8: Water | ChemComp-HOH / | |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: 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. THERE IS NO ssDNA BOUND TO BCDX2 IN THIS STRUCTURE Entity ID: #1-#4 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||||||||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pBIG-BCDX2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: 25 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5 mM ADP.AlFx (0.5 mM ADP, 0.5 mM AlCl3, 10 mM NaF), 0.25 mM TCEP, 0.00075% Tween20, 0.075 mM CHAPSO | ||||||||||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||||||||||||||||
Specimen support | Details: 45 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.78 sec. / Electron dose: 53.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 35305 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 25886382 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1371033 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 50.38 / Protocol: RIGID BODY FIT / Space: REAL Details: PDB-8OUY was initially docked into the postprocessed map using ChimeraX 1.4, and iteratively real space refined with Phenix and manually modified using COOT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 8OUY Accession code: 8OUY / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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