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Yorodumi- EMDB-2780: Electron microscopy of human BRCA2 tumour suppressor bound to hum... -
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-Basic information
Entry | Database: EMDB / ID: EMD-2780 | |||||||||
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Title | Electron microscopy of human BRCA2 tumour suppressor bound to human RAD51 recombinase | |||||||||
Map data | Reconstruction of full-length human BRCA2 in complex with human RAD51 recombinase | |||||||||
Sample |
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Keywords | recombination mediator / tumour suppressor / recombinase / DNA double-strand break repair / homologous recombination / negative stain | |||||||||
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) / centrosome duplication / nuclear ubiquitin ligase complex / double-strand break repair involved in meiotic recombination / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / DNA recombinase assembly / telomere maintenance via recombination / histone H4 acetyltransferase activity / histone H3 acetyltransferase activity / regulation of DNA damage checkpoint / mitotic recombination / DNA strand invasion / Impaired BRCA2 binding to PALB2 / HDR through MMEJ (alt-NHEJ) / DNA strand exchange activity / gamma-tubulin binding / reciprocal meiotic recombination / single-stranded DNA helicase activity / 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 / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / response to UV-C / oocyte maturation / Resolution of D-loop Structures through Holliday Junction Intermediates / 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 / HDR through Single Strand Annealing (SSA) / hematopoietic stem cell proliferation / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / condensed chromosome / female gonad development / DNA unwinding involved in DNA replication / replication fork processing / male meiosis I / Transcriptional Regulation by E2F6 / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / condensed nuclear chromosome / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / ATP-dependent activity, acting on DNA / meiotic cell cycle / male germ cell nucleus / interstrand cross-link repair / DNA polymerase binding / positive regulation of mitotic cell cycle / regulation of cytokinesis / Meiotic recombination / secretory granule / cellular response to ionizing radiation / nucleotide-excision repair / response to gamma radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / brain development / HDR through Homologous Recombination (HRR) / PML body / cellular senescence / double-strand break repair / double-stranded DNA binding / site of double-strand break / single-stranded DNA binding / chromosome, telomeric region / spermatogenesis / protease binding / DNA recombination / mitochondrial matrix / DNA repair / chromatin binding / centrosome / DNA damage response / positive regulation of DNA-templated transcription / chromatin / regulation of DNA-templated transcription / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 19.5 Å | |||||||||
Authors | Shahid T / Soroka J / Kong EH / Malivert L / McIlwraith MJ / Pape T / West SC / Zhang X | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2014 Title: Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor. Authors: Taha Shahid / Joanna Soroka / Eric Kong / Laurent Malivert / Michael J McIlwraith / Tillman Pape / Stephen C West / Xiaodong Zhang / Abstract: Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, has a key role in the repair of DNA double-strand breaks and interstrand ...Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, has a key role in the repair of DNA double-strand breaks and interstrand cross-links by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full-length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional EM reconstructions revealed that BRCA2 exists as a dimer and that two oppositely oriented sets of RAD51 molecules bind the dimer. Single-stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous-recombinational repair. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2780.map.gz | 706.8 KB | EMDB map data format | |
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Header (meta data) | emd-2780-v30.xml emd-2780.xml | 17.3 KB 17.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_2780_fsc.xml | 5.4 KB | Display | FSC data file |
Images | emd_2780.png emd_2780_1.png | 62.2 KB 39.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2780 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2780 | HTTPS FTP |
-Validation report
Summary document | emd_2780_validation.pdf.gz | 222.9 KB | Display | EMDB validaton report |
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Full document | emd_2780_full_validation.pdf.gz | 222 KB | Display | |
Data in XML | emd_2780_validation.xml.gz | 8.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2780 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2780 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2780.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of full-length human BRCA2 in complex with human RAD51 recombinase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.52 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Full-length human BRCA2 protein complexed with RAD51 recombinase
Entire | Name: Full-length human BRCA2 protein complexed with RAD51 recombinase |
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Components |
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-Supramolecule #1000: Full-length human BRCA2 protein complexed with RAD51 recombinase
Supramolecule | Name: Full-length human BRCA2 protein complexed with RAD51 recombinase type: sample / ID: 1000 / Oligomeric state: Dimer / Number unique components: 2 |
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Molecular weight | Experimental: 1.2 MDa / Theoretical: 1.1 MDa / Method: Scanning transmission electron microscopy |
-Macromolecule #1: Breast cancer type 2 susceptibility protein
Macromolecule | Name: Breast cancer type 2 susceptibility protein / type: protein_or_peptide / ID: 1 / Name.synonym: BRCA2 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus |
Molecular weight | Theoretical: 384 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HeLa |
Sequence | UniProtKB: Breast cancer type 2 susceptibility protein / InterPro: Breast cancer type 2 susceptibility protein |
-Macromolecule #2: DNA repair protein RAD51 homolog 1
Macromolecule | Name: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 2 / Name.synonym: HsRAD51 / Number of copies: 8 / Oligomeric state: Dimer of tetramers / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus |
Molecular weight | Theoretical: 37 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: DNA repair protein RAD51 homolog 1 |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Staining | Type: NEGATIVE Details: Grid with adsorbed protein was washed twice with water and negatively stained with 2% uranyl acetate solution before being blotted and air dried. |
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Grid | Details: Copper Quantifoil R2/2 holey carbon grids coated with continuous carbon, glow discharged in air. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy #1
Microscopy ID | 1 |
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Microscope | FEI/PHILIPS CM200FEG |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. |
Date | Dec 14, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Electron microscopy #2
Microscopy ID | 2 |
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Microscope | FEI/PHILIPS CM200FEG |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. |
Date | Dec 19, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Electron microscopy #3
Microscopy ID | 3 |
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Microscope | FEI/PHILIPS CM200FEG |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. |
Date | Dec 20, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Electron microscopy #4
Microscopy ID | 4 |
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Microscope | FEI/PHILIPS CM200FEG |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. |
Date | Mar 11, 2013 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Electron microscopy #5
Microscopy ID | 5 |
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Microscope | FEI/PHILIPS CM200FEG |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. |
Date | Mar 14, 2013 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: UCSF Chimera |
Details | The yeast Rad51 filament structure was interactively fitted into into the map (with the aid of antibody-labelling) and the fit refined in UCSF Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient |