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- EMDB-21998: EM structure of human tumor suppressor bound to human DSS1 protei... -

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Basic information

Entry
Database: EMDB / ID: EMD-21998
TitleEM structure of human tumor suppressor bound to human DSS1 protein and ssDNA with crosslinking
Map dataHuman BRCA2-DSS1-ssDNA complex with crosslinking
Sample
  • Complex: BRCA2-DSS1-ssDNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.6 Å
AuthorsLe HP / Ma X / Vaquero J / Brinkmeyer M / Guo F / Heyer W-D / Liu J
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA187561 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM58015 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA92267 United States
CitationJournal: Nucleic Acids Res / Year: 2020
Title: DSS1 and ssDNA regulate oligomerization of BRCA2.
Authors: Hang Phuong Le / Xiaoyan Ma / Jorge Vaquero / Megan Brinkmeyer / Fei Guo / Wolf-Dietrich Heyer / Jie Liu /
Abstract: The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner ...The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.
History
DepositionMay 20, 2020-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0063
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0063
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21998.png

Downloads & links

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Map

FileDownload / File: emd_21998.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman BRCA2-DSS1-ssDNA complex with crosslinking
Voxel sizeX=Y=Z: 1.48 Å
Density
Contour LevelBy AUTHOR: 0.0063 / Movie #1: 0.0063
Minimum - Maximum-0.009578008 - 0.06953412
Average (Standard dev.)0.0001805619 (±0.0017589984)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 378.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.481.481.48
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z378.880378.880378.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.0100.0700.000

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Supplemental data

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Sample components

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Entire : BRCA2-DSS1-ssDNA

EntireName: BRCA2-DSS1-ssDNA
Components
  • Complex: BRCA2-DSS1-ssDNA

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Supramolecule #1: BRCA2-DSS1-ssDNA

SupramoleculeName: BRCA2-DSS1-ssDNA / type: complex / ID: 1 / Parent: 0
Details: Full-length human tumor suppressor BRCA2 protein in complex with DSS1 and dT100 oligo with crosslinking
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293
Molecular weightExperimental: 474 kDa/nm

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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

BufferpH: 7.5
StainingType: NEGATIVE / Material: 2% uranyl acetate
Details: BRCA2 complex was applied to glow-discharged continuous carbon copper grids and incubated for 2 min. Grids were washed briefly with 5 mM Mg(Acet)2 for three times to remove buffer, sucrose, ...Details: BRCA2 complex was applied to glow-discharged continuous carbon copper grids and incubated for 2 min. Grids were washed briefly with 5 mM Mg(Acet)2 for three times to remove buffer, sucrose, and glycerol. Samples were then stained with 2% uranyl acetate, blotted with filter paper, and air dried.
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: JEOL
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Digitization - Dimensions - Width: 5120 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Sampling interval: 6.4 µm / Number grids imaged: 1 / Number real images: 1359 / Average exposure time: 1.0 sec. / Average electron dose: 37.5 e/Å2

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Image processing

Particle selectionNumber selected: 80985
CTF correctionSoftware - Name: SPHIRE (ver. 1.2)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.2)
Final 3D classificationSoftware - Name: SPHIRE (ver. 1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 14.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPHIRE (ver. 1.2) / Number images used: 18429
FSC plot (resolution estimation)

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