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- PDB-8pbd: RAD51 filament on dsDNA bound by the BRCA2 c-terminus -

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Basic information

Entry
Database: PDB / ID: 8pbd
TitleRAD51 filament on dsDNA bound by the BRCA2 c-terminus
Components
  • Breast cancer type 2 susceptibility protein
  • DNA repair protein RAD51 homolog 1
  • DNA strand 1
  • DNA strand 2
KeywordsRECOMBINATION / RAD51 / BRCA2 / Filament / Complex
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 / establishment of protein localization to telomere / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening ...BRCA2-MAGE-D1 complex / negative regulation of mammary gland epithelial cell proliferation / presynaptic intermediate filament cytoskeleton / response to glucoside / establishment of protein localization to telomere / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / 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) / histone H4 acetyltransferase activity / DNA strand exchange activity / histone H3 acetyltransferase activity / replication-born double-strand break repair via sister chromatid exchange / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / telomere maintenance via recombination / gamma-tubulin binding / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / oocyte maturation / DNA repair complex / reciprocal meiotic recombination / response to UV-C / inner cell mass cell proliferation / 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 / HDR through Single Strand Annealing (SSA) / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / nuclear chromosome / female gonad development / hematopoietic stem cell proliferation / Impaired BRCA2 binding to RAD51 / 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 / condensed nuclear chromosome / regulation of cytokinesis / response to gamma radiation / cellular response to ionizing radiation / meiotic cell cycle / male germ cell nucleus / nucleotide-excision repair / DNA damage response, signal transduction by p53 class mediator / cellular response to gamma radiation / double-strand break repair via homologous recombination / PML body / brain development / HDR through Homologous Recombination (HRR) / response to toxic substance / Meiotic recombination / cellular senescence / double-strand break repair / single-stranded DNA binding / site of double-strand break / protease binding / double-stranded DNA binding / spermatogenesis / DNA recombination / chromosome, telomeric region / mitochondrial matrix / response to xenobiotic stimulus / DNA repair / DNA damage response / centrosome / 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
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Breast cancer type 2 susceptibility protein / DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsAppleby, R. / Pellegrini, L.
Funding support United Kingdom, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release

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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 1
B: DNA repair protein RAD51 homolog 1
C: DNA repair protein RAD51 homolog 1
D: DNA repair protein RAD51 homolog 1
E: DNA repair protein RAD51 homolog 1
F: DNA repair protein RAD51 homolog 1
G: DNA repair protein RAD51 homolog 1
H: DNA repair protein RAD51 homolog 1
I: DNA repair protein RAD51 homolog 1
J: DNA repair protein RAD51 homolog 1
K: Breast cancer type 2 susceptibility protein
L: Breast cancer type 2 susceptibility protein
M: Breast cancer type 2 susceptibility protein
N: Breast cancer type 2 susceptibility protein
O: Breast cancer type 2 susceptibility protein
P: Breast cancer type 2 susceptibility protein
Q: Breast cancer type 2 susceptibility protein
R: Breast cancer type 2 susceptibility protein
S: Breast cancer type 2 susceptibility protein
T: DNA strand 1
U: DNA strand 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)441,91351
Polymers436,04021
Non-polymers5,87330
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules TU

#3: DNA chain DNA strand 1


Mass: 8699.608 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA strand 2


Mass: 7898.048 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Protein/peptide , 2 types, 19 molecules ABCDEFGHIJKLMNOPQRS

#1: Protein
DNA repair protein RAD51 homolog 1 / HsRAD51 / hRAD51 / RAD51 homolog A


Mass: 37009.125 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli (E. coli) / References: UniProt: Q06609
#2: Protein/peptide
Breast cancer type 2 susceptibility protein / Fanconi anemia group D1 protein


Mass: 5483.415 Da / Num. of mol.: 9 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P51587

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Non-polymers , 2 types, 30 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1RAD51 filament formed on dsDNA and bound with a peptide corresponding to the BRCA2 c-terminusCOMPLEX#1-#40MULTIPLE SOURCES
2DNA repair protein RAD51 homolog 1COMPLEX#11RECOMBINANTRAD51 was recombinantly purified BRCA2 c-terminus peptide was synthesised 27-mer DNA strands were synthesised and annealed
3Breast cancer type 2 susceptibility proteinCOMPLEX#21RECOMBINANT
4DNA strand 1 and 2COMPLEX#3-#41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
22Escherichia coli (E. coli)562pMBP4-RAD51
33synthetic construct (others)32630
44synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 52.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4CTFFIND4.1.5CTF correction
7Coot0.9.8.5model fitting
9PHENIXdev-4707model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 56 ° / Axial rise/subunit: 15.8 Å / Axial symmetry: C1
3D reconstructionResolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL

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