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- EMDB-17928: Structure of BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosome... -

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Basic information

Entry
Database: EMDB / ID: EMD-17928
TitleStructure of BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes (Map 2)
Map data
Sample
  • Complex: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes
    • Complex: DNA, Histones and Polyubiquitin-B
    • Complex: BRCA1 associated RING domain 1
KeywordsBRCA1-BARD1 / chromatin recognition / nucleosomes / ubiquitin / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human) / Felis catus (domestic cat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsFoglizzo M / Burdett H / Wilson MD / Zeqiraj E
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/T029471/1 United Kingdom
Wellcome Trust222531/Z/21/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Wellcome Trust221524/Z/20/Z United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2023
Title: BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes.
Authors: Hayden Burdett / Martina Foglizzo / Laura J Musgrove / Dhananjay Kumar / Gillian Clifford / Lisa J Campbell / George R Heath / Elton Zeqiraj / Marcus D Wilson /
Abstract: Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with ...Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural and single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.
History
DepositionJul 16, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17928.png

Downloads & links

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Map

FileDownload / File: emd_17928.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 320 pix.
= 275.2 Å		0.86 Å/pix.
x 320 pix.
= 275.2 Å		0.86 Å/pix.
x 320 pix.
= 275.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.263
Minimum - Maximum-1.4423823 - 2.4594595
Average (Standard dev.)0.006351845 (±0.061098855)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17928_msk_1.map
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Additional map: #1

Fileemd_17928_additional_1.map
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Additional map: #2

Fileemd_17928_additional_2.map
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Half map: #2

Fileemd_17928_half_map_1.map
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Half map: #1

Fileemd_17928_half_map_2.map
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Sample components

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Entire : BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes

EntireName: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes
Components
  • Complex: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes
    • Complex: DNA, Histones and Polyubiquitin-B
    • Complex: BRCA1 associated RING domain 1

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Supramolecule #1: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes

SupramoleculeName: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Molecular weightTheoretical: 246 KDa

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Supramolecule #2: DNA, Histones and Polyubiquitin-B

SupramoleculeName: DNA, Histones and Polyubiquitin-B / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #7
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: BRCA1 associated RING domain 1

SupramoleculeName: BRCA1 associated RING domain 1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Felis catus (domestic cat)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
50.0 mMNaClsodium chloride

Details: 20 mM HEPES pH 7.5, 50 mM NaCl and 1 mM DTT
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
Details: Quantifoil R3.5/1 200-mesh grids (Quantifoil Micro Tools GmbH) were glow-discharged for 30 s at 40 mA using a GloQube (Quorum) glow discharge unit
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force = 0 N blot time = 8 s.
DetailsPurified cat BRCA1dExon11-FL BARD1 (at 3 uM) was incubated with H2AKc15ub mono-nucleosomes (at 1.5 uM) for 1 h on ice before cryo-EM grids preparation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 16015 / Average exposure time: 5.0 sec. / Average electron dose: 36.37 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7204662
Startup modelType of model: OTHER
Details: The start up model was generated directly from this dataset, following 2D Classification and Ab Initio Reconstruction in cryoSPARC 3.2.0
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 165464
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.1)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.0.1)

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