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- EMDB-17585: RAD51 filament on dsDNA bound by the BRCA2 c-terminus -

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Basic information

Entry
Database: EMDB / ID: EMD-17585
TitleRAD51 filament on dsDNA bound by the BRCA2 c-terminus
Map dataRAD51 filament on dsDNA bound by the BRCA2 c-terminus
Sample
  • Complex: RAD51 filament formed on dsDNA 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 strand 1 and 2
      • DNA: DNA strand 1
      • DNA: DNA strand 2
  • 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 / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / establishment of protein localization to telomere / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / Impaired BRCA2 translocation to the nucleus ...BRCA2-MAGE-D1 complex / negative regulation of mammary gland epithelial cell proliferation / presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / establishment of protein localization to telomere / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / Impaired BRCA2 translocation to the nucleus / Impaired BRCA2 binding to SEM1 (DSS1) / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / telomere maintenance via recombination / DNA recombinase assembly / histone H4 acetyltransferase activity / histone H3 acetyltransferase activity / regulation of DNA damage checkpoint / mitotic recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion / HDR through MMEJ (alt-NHEJ) / gamma-tubulin binding / DNA strand exchange activity / reciprocal meiotic recombination / DNA repair complex / 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 / oocyte maturation / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / response to UV-C / Resolution of D-loop Structures through Holliday Junction Intermediates / single-stranded DNA helicase activity / inner cell mass cell proliferation / ATP-dependent DNA damage sensor activity / DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / hematopoietic stem cell proliferation / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / female gonad development / replication fork processing / male meiosis I / Transcriptional Regulation by E2F6 / DNA unwinding involved in DNA replication / Presynaptic phase of homologous DNA pairing and strand exchange / centrosome duplication / response to X-ray / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / positive regulation of mitotic cell cycle / meiotic cell cycle / regulation of cytokinesis / condensed nuclear chromosome / secretory granule / male germ cell nucleus / cellular response to ionizing radiation / nucleotide-excision repair / response to gamma radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / brain development / PML body / Meiotic recombination / cellular senescence / double-strand break repair / site of double-strand break / single-stranded DNA binding / spermatogenesis / double-stranded DNA binding / DNA recombination / protease binding / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / regulation of DNA-templated transcription / chromatin / nucleolus / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding
Similarity search - Function
: / 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 / Tower ...: / 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 / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / 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: 2.83 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_17585.png

Downloads & links

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Map

FileDownload / File: emd_17585.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRAD51 filament on dsDNA bound by the BRCA2 c-terminus
Voxel sizeX=Y=Z: 1.304 Å
Density
Contour LevelBy AUTHOR: 0.191
Minimum - Maximum-1.5603029 - 2.7862973
Average (Standard dev.)0.000000000000364 (±0.07160315)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=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_17585_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_17585_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 dsDNA and bound with a peptide correspon...

EntireName: RAD51 filament formed on dsDNA and bound with a peptide corresponding to the BRCA2 c-terminus
Components
  • Complex: RAD51 filament formed on dsDNA 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 strand 1 and 2
      • DNA: DNA strand 1
      • DNA: DNA strand 2
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM ION

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

SupramoleculeName: RAD51 filament formed on dsDNA and bound with a peptide corresponding to the BRCA2 c-terminus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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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 27-mer DNA strands were synthesised and annealed
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 strand 1 and 2

SupramoleculeName: DNA strand 1 and 2 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
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: 10 / 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: 9 / 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 strand 1

MacromoleculeName: DNA strand 1 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.699608 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)

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Macromolecule #4: DNA strand 2

MacromoleculeName: DNA strand 2 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.898048 KDa
SequenceString:
(DT)(DC)(DC)(DT)(DC)(DC)(DT)(DC)(DC)(DT) (DC)(DC)(DT)(DC)(DC)(DT)(DC)(DC)(DT)(DC) (DC)(DT)(DC)(DC)(DT)(DC)(DC)

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 10 / 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 #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 20 / 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
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: 15.8 Å
Applied symmetry - Helical parameters - Δ&Phi: 56.0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.83 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-8pbd:
RAD51 filament on dsDNA bound by the BRCA2 c-terminus

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