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- EMDB-2780: Electron microscopy of human BRCA2 tumour suppressor bound to hum... -

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Basic information

Entry
Database: EMDB / ID: EMD-2780
TitleElectron microscopy of human BRCA2 tumour suppressor bound to human RAD51 recombinase
Map dataReconstruction of full-length human BRCA2 in complex with human RAD51 recombinase
Sample
  • Sample: Full-length human BRCA2 protein complexed with RAD51 recombinase
  • Protein or peptide: Breast cancer type 2 susceptibility protein
  • Protein or peptide: DNA repair protein RAD51 homolog 1
Keywordsrecombination 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 / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / establishment of protein localization to telomere / telomere maintenance via telomere lengthening / positive regulation of DNA ligation ...BRCA2-MAGE-D1 complex / negative regulation of mammary gland epithelial cell proliferation / presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / 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) / mitotic recombination / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / DNA strand invasion / replication-born double-strand break repair via sister chromatid exchange / histone H3 acetyltransferase activity / histone H4 acetyltransferase activity / cellular response to hydroxyurea / DNA strand exchange activity / lateral element / telomere maintenance via recombination / regulation of DNA damage checkpoint / HDR through MMEJ (alt-NHEJ) / oocyte maturation / Impaired BRCA2 binding to PALB2 / gamma-tubulin binding / single-stranded DNA helicase 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 / Homologous DNA Pairing and Strand Exchange / response to UV-C / 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) / inner cell mass cell proliferation / Impaired BRCA2 binding to RAD51 / hematopoietic stem cell proliferation / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / nuclear chromosome / replication fork processing / DNA unwinding involved in DNA replication / female gonad development / male meiosis I / Transcriptional Regulation by E2F6 / DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / 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 / protein autoubiquitination / DNA polymerase binding / condensed chromosome / positive regulation of mitotic cell cycle / meiotic cell cycle / regulation of cytokinesis / condensed nuclear chromosome / male germ cell nucleus / secretory granule / cellular response to ionizing radiation / nucleotide-excision repair / response to gamma radiation / double-strand break repair via homologous recombination / regulation of protein phosphorylation / brain development / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / double-strand break repair / cellular senescence / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / spermatogenesis / DNA recombination / protease binding / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / regulation of DNA-templated transcription / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP 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 / Breast cancer type 2 susceptibility protein / 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)
Methodsingle particle reconstruction / negative staining / Resolution: 19.5 Å
AuthorsShahid T / Soroka J / Kong EH / Malivert L / McIlwraith MJ / Pape T / West SC / Zhang X
CitationJournal: 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
DepositionSep 3, 2014-
Header (metadata) releaseSep 17, 2014-
Map releaseOct 8, 2014-
UpdateOct 8, 2014-
Current statusOct 8, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2780.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of full-length human BRCA2 in complex with human RAD51 recombinase
Voxel sizeX=Y=Z: 3.52 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.31503955 - 0.71593612
Average (Standard dev.)0.00124616 (±0.02884739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.523.523.52
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z450.560450.560450.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.3150.7160.001

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Supplemental data

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Sample components

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Entire : Full-length human BRCA2 protein complexed with RAD51 recombinase

EntireName: Full-length human BRCA2 protein complexed with RAD51 recombinase
Components
  • Sample: Full-length human BRCA2 protein complexed with RAD51 recombinase
  • Protein or peptide: Breast cancer type 2 susceptibility protein
  • Protein or peptide: DNA repair protein RAD51 homolog 1

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Supramolecule #1000: Full-length human BRCA2 protein complexed with RAD51 recombinase

SupramoleculeName: Full-length human BRCA2 protein complexed with RAD51 recombinase
type: sample / ID: 1000 / Oligomeric state: Dimer / Number unique components: 2
Molecular weightExperimental: 1.2 MDa / Theoretical: 1.1 MDa / Method: Scanning transmission electron microscopy

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Macromolecule #1: Breast cancer type 2 susceptibility protein

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Molecular weightTheoretical: 384 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HeLa
SequenceUniProtKB: Breast cancer type 2 susceptibility protein / InterPro: Breast cancer type 2 susceptibility protein

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Macromolecule #2: DNA repair protein RAD51 homolog 1

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Molecular weightTheoretical: 37 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: DNA repair protein RAD51 homolog 1

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: 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.
GridDetails: Copper Quantifoil R2/2 holey carbon grids coated with continuous carbon, glow discharged in air.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy #1

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Microscopy ID1
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateDec 14, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16

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Electron microscopy #2

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Microscopy ID2
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateDec 19, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16

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Electron microscopy #3

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Microscopy ID3
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateDec 20, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16

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Electron microscopy #4

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Microscopy ID4
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateMar 11, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16

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Electron microscopy #5

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Microscopy ID5
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateMar 14, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 40 e/Å2 / Bits/pixel: 16

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Image processing

Final two d classificationNumber classes: 553
Final angle assignmentDetails: IMAGIC: 0 < Beta < 180 degrees, -90 < Gamma < 90 degrees
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.5 Å / Resolution method: OTHER / Software - Name: IMAGIC-5, Tigris
Details: Final maps were calculated after a round of projection matching.
Number images used: 6877
DetailsThe particles were selected manually in EMAN2.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: UCSF Chimera
DetailsThe yeast Rad51 filament structure was interactively fitted into into the map (with the aid of antibody-labelling) and the fit refined in UCSF Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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