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- EMDB-17207: Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour s... -

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Entry
Database: EMDB / ID: EMD-17207
TitleStructure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor
Map datadeepEMhancer_map
Sample
  • Complex: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)
KeywordsComplex / ssDNA-binding / ATPase / RAD51 / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGreenhough LA / Liang CC / West SC
Funding support United Kingdom, European Union, 5 items
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
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.
#1: Journal: Nature / Year: 2023
Title: Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor
Authors: Greenhough LA / Liang CC / Belan O / Kunzelmann S / Maslen S / Rodrigo-Brenni MC / Anand R / Skehel M / Boulton SJ / West SC
History
DepositionApr 25, 2023-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17207.png

Downloads & links

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Map

FileDownload / File: emd_17207.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer_map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 300 pix.
= 306. Å		1.02 Å/pix.
x 300 pix.
= 306. Å		1.02 Å/pix.
x 300 pix.
= 306. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.02 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0144
Minimum - Maximum-0.024954095 - 2.0900557
Average (Standard dev.)0.000605656 (±0.01688938)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: refine3D map

Fileemd_17207_additional_1.map
Annotationrefine3D_map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_17207_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_17207_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)

EntireName: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)
Components
  • Complex: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)

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Supramolecule #1: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)

SupramoleculeName: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) with 30 nt ssDNA (random sequence)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Recombinant BCDX2 complexes purified from Sf9 insect cells, vitrified in the presence of ADP.AlFx
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHydroxyethylpiperazine
100.0 mMNaClSodium chlorideSodium chloride
2.5 mMMgCl2Magnesium chloride
0.5 mMADPAdenosine diphosphateAdenosine diphoshpate
0.5 mMAlCl3Aluminium chloride
10.0 mMNaFSodium fluoride
0.25 mMTCEPTris(2-carboxyethyl)phosphine
0.00075 %Tween20Polysorbate 20
0.075 mMCHAPSOCHAPS detergent3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate

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
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 45 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 35305 / Average exposure time: 2.78 sec. / Average electron dose: 53.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 25886382
Startup modelType of model: EMDB MAP
EMDB ID:

17205


Details: Map of BCDX2 consensus refinement
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 30 / Avg.num./class: 156505 / Software - Name: RELION (ver. 4)
Details: Signal subtraction of ssDNA binding site, followed by focussed 3D classification without alignment
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 241626
FSC plot (resolution estimation)

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