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- EMDB-17584: RAD51 filament on ssDNA bound by the BRCA2 c-terminus -

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Basic information

Entry
Database: EMDB / ID: EMD-17584
TitleRAD51 filament on ssDNA bound by the BRCA2 c-terminus
Map dataCryoEM map of a RAD51 filament on ssDNA bound with the BRCA2 c-terminus
Sample
  • Complex: RAD51 filament formed on ssDNA and bound with a peptide corresponding to the BRCA2 c-terminus
    • Complex: DNA repair protein RAD51 homolog 1
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: Breast cancer type 2 susceptibility protein
      • Protein or peptide: Breast cancer type 2 susceptibility protein
    • Complex: DNA (30-MER)
      • DNA: DNA (30-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM ION
KeywordsRAD51 / BRCA2 / Filament / Complex / RECOMBINATION
Function / homology
Function and homology information


BRCA2-MAGE-D1 complex / negative regulation of mammary gland epithelial cell proliferation / presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / establishment of protein localization to telomere / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / cellular response to camptothecin / telomere maintenance via telomere lengthening ...BRCA2-MAGE-D1 complex / negative regulation of mammary gland epithelial cell proliferation / presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / establishment of protein localization to telomere / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / cellular response to camptothecin / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to cisplatin / DNA strand invasion / cellular response to hydroxyurea / mitotic recombination / Impaired BRCA2 translocation to the nucleus / Impaired BRCA2 binding to SEM1 (DSS1) / DNA strand exchange activity / histone H4 acetyltransferase activity / replication-born double-strand break repair via sister chromatid exchange / histone H3 acetyltransferase activity / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / HDR through MMEJ (alt-NHEJ) / gamma-tubulin binding / reciprocal meiotic recombination / single-stranded DNA helicase activity / DNA repair complex / oocyte maturation / response to UV-C / Homologous DNA Pairing and Strand Exchange / 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 / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / inner cell mass cell proliferation / HDR through Single Strand Annealing (SSA) / regulation of double-strand break repair via homologous recombination / ATP-dependent DNA damage sensor activity / nuclear chromosome / Impaired BRCA2 binding to RAD51 / female gonad development / hematopoietic stem cell proliferation / Transcriptional Regulation by E2F6 / male meiosis I / replication fork processing / centrosome duplication / Presynaptic phase of homologous DNA pairing and strand exchange / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / condensed chromosome / DNA polymerase binding / positive regulation of mitotic cell cycle / secretory granule / male germ cell nucleus / regulation of cytokinesis / condensed nuclear chromosome / meiotic cell cycle / cellular response to ionizing radiation / response to gamma radiation / nucleotide-excision repair / DNA damage response, signal transduction by p53 class mediator / double-strand break repair via homologous recombination / cellular response to gamma radiation / brain development / PML body / HDR through Homologous Recombination (HRR) / Meiotic recombination / response to toxic substance / cellular senescence / double-strand break repair / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / protease binding / spermatogenesis / DNA recombination / chromosome, telomeric region / mitochondrial matrix / response to xenobiotic stimulus / DNA repair / centrosome / DNA damage response / chromatin binding / regulation of DNA-templated transcription / chromatin / positive regulation of DNA-templated transcription / nucleolus / perinuclear region of cytoplasm / enzyme binding / protein-containing complex / ATP hydrolysis activity / mitochondrion / nucleoplasm
Similarity search - Function
BRCA2, OB3 / Tower domain / Breast cancer type 2 susceptibility protein, helical domain / BRCA2 helical domain superfamily / : / : / BRCA2, oligonucleotide/oligosaccharide-binding, domain 3 / Tower / BRCA2, helical / BRCA2, OB2 ...BRCA2, OB3 / Tower domain / Breast cancer type 2 susceptibility protein, helical domain / BRCA2 helical domain superfamily / : / : / BRCA2, oligonucleotide/oligosaccharide-binding, domain 3 / Tower / BRCA2, helical / BRCA2, OB2 / BRCA2 TR2 domain / Tower / BRCA2 repeat / BRCA2, OB1 / Breast cancer type 2 susceptibility protein / BRCA2 repeat / BRCA2, oligonucleotide/oligosaccharide-binding, domain 1 / BRCA2 repeat profile. / DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Breast cancer type 2 susceptibility protein / DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsAppleby R / Pellegrini L
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/ United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)2271086 United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for stabilisation of the RAD51 nucleoprotein filament by BRCA2.
Authors: Robert Appleby / Luay Joudeh / Katie Cobbett / Luca Pellegrini /
Abstract: The BRCA2 tumour suppressor protein preserves genomic integrity via interactions with the DNA-strand exchange RAD51 protein in homology-directed repair. The RAD51-binding TR2 motif at the BRCA2 C- ...The BRCA2 tumour suppressor protein preserves genomic integrity via interactions with the DNA-strand exchange RAD51 protein in homology-directed repair. The RAD51-binding TR2 motif at the BRCA2 C-terminus is essential for protection and restart of stalled replication forks. Biochemical evidence shows that TR2 recognises filamentous RAD51, but existing models of TR2 binding to RAD51 lack a structural basis. Here we used cryo-electron microscopy and structure-guided mutagenesis to elucidate the mechanism of TR2 binding to nucleoprotein filaments of human RAD51. We find that TR2 binds across the protomer interface in the filament, acting as a brace for adjacent RAD51 molecules. TR2 targets an acidic-patch motif on human RAD51 that serves as a recruitment hub in fission yeast Rad51 for recombination mediators Rad52 and Rad55-Rad57. Our findings provide a structural rationale for RAD51 filament stabilisation by BRCA2 and reveal a common recruitment mechanism of recombination mediators to the RAD51 filament.
History
DepositionJun 9, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17584.png

Downloads & links

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Map

FileDownload / File: emd_17584.map.gz / Format: CCP4 / Size: 5.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of a RAD51 filament on ssDNA bound with the BRCA2 c-terminus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.3 Å/pix.
x 83 pix.
= 108.232 Å		1.3 Å/pix.
x 84 pix.
= 109.536 Å		1.3 Å/pix.
x 200 pix.
= 260.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.304 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.166
Minimum - Maximum-2.1750417 - 4.0552206
Average (Standard dev.)-0.000000000004897 (±0.21122044)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin59059
Dimensions8420083
Spacing8384200
CellA: 108.232 Å / B: 109.536 Å / C: 260.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map 1

Fileemd_17584_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_17584_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 : RAD51 filament formed on ssDNA and bound with a peptide correspon...

EntireName: RAD51 filament formed on ssDNA and bound with a peptide corresponding to the BRCA2 c-terminus
Components
  • Complex: RAD51 filament formed on ssDNA and bound with a peptide corresponding to the BRCA2 c-terminus
    • Complex: DNA repair protein RAD51 homolog 1
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: Breast cancer type 2 susceptibility protein
      • Protein or peptide: Breast cancer type 2 susceptibility protein
    • Complex: DNA (30-MER)
      • DNA: DNA (30-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM ION

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Supramolecule #1: RAD51 filament formed on ssDNA and bound with a peptide correspon...

SupramoleculeName: RAD51 filament formed on ssDNA and bound with a peptide corresponding to the BRCA2 c-terminus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: DNA repair protein RAD51 homolog 1

SupramoleculeName: DNA repair protein RAD51 homolog 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: RAD51 was recombinantly purified BRCA2 c-terminus peptide was synthesised 30-mer ssDNA was synthesised
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Breast cancer type 2 susceptibility protein

SupramoleculeName: Breast cancer type 2 susceptibility protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA (30-MER)

SupramoleculeName: DNA (30-MER) / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DNA repair protein RAD51 homolog 1

MacromoleculeName: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 11 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.009125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...String:
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQVDGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD

UniProtKB: DNA repair protein RAD51 homolog 1

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Macromolecule #2: Breast cancer type 2 susceptibility protein

MacromoleculeName: Breast cancer type 2 susceptibility protein / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.483415 KDa
SequenceString:
DDQKNCKKRR ALDFLSRLPL PPPVSPICTF VSPAAQKAFQ PPRSCGTKY

UniProtKB: Breast cancer type 2 susceptibility protein

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Macromolecule #3: DNA (30-MER)

MacromoleculeName: DNA (30-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.671227 KDa
SequenceString:
(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG) (DG)(DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG) (DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG) (DA)

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 11 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 22 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridMaterial: GOLD
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 16.1 Å
Applied symmetry - Helical parameters - Δ&Phi: 56.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 1
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8pbc:
RAD51 filament on ssDNA bound by the BRCA2 c-terminus

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