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- EMDB-16870: Low-resolution structure of BRCA1dExon11-FL BARD1 (open state) -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-16870
TitleLow-resolution structure of BRCA1dExon11-FL BARD1 (open state)
Map data
Sample
  • Complex: Heterodimeric complex of BRCA1dExon11 and FL BARD1
KeywordsProtein complex / heterodimer / ubiquitin / E3 ligase / chromatin association / LIGASE
Biological speciesFelis catus (domestic cat)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.4 Å
AuthorsFoglizzo M / 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
DepositionMar 19, 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_16870.png

Downloads & links

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Map

FileDownload / File: emd_16870.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 260 pix.
= 276.9 Å		1.07 Å/pix.
x 260 pix.
= 276.9 Å		1.07 Å/pix.
x 260 pix.
= 276.9 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0113
Minimum - Maximum-0.007314062 - 0.06339154
Average (Standard dev.)0.00010515639 (±0.0026844023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 276.90002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16870_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16870_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Heterodimeric complex of BRCA1dExon11 and FL BARD1

EntireName: Heterodimeric complex of BRCA1dExon11 and FL BARD1
Components
  • Complex: Heterodimeric complex of BRCA1dExon11 and FL BARD1

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Supramolecule #1: Heterodimeric complex of BRCA1dExon11 and FL BARD1

SupramoleculeName: Heterodimeric complex of BRCA1dExon11 and FL BARD1 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Felis catus (domestic cat)
Molecular weightTheoretical: 175 KDa

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
300.0 mMNaClsodium chloride

Details: 25 mM HEPES pH 7.5, 300 mM NaCl, 5% (v/v) glycerol and 1 mM TCEP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Details: UltrAuFoil R1.2/1.3 300-mesh gold grids (Quantifoil Micro Tools GmbH) were cleaned via indirect plasma using a Tergeo EM plasma cleaner (Pie Scientific) at 15 W for 1 min, and with flow ...Details: UltrAuFoil R1.2/1.3 300-mesh gold grids (Quantifoil Micro Tools GmbH) were cleaned via indirect plasma using a Tergeo EM plasma cleaner (Pie Scientific) at 15 W for 1 min, and with flow rates for nitrogen, oxygen and argon gasses of 20.0, 19.8 and 29.0 sscm respectively
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Vitrification was carried out by using a blot force of 6 N and a blot time of 6 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 227 / Average exposure time: 1.7 sec. / Average electron dose: 73.0 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: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
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: 117630
Details: Initially, 1,368 particles were manually picked and used to train crYOLO v1.6.1. This trained model was used for picking on all 227 movies, resulting in 117,630 particles
Startup modelType of model: OTHER
Details: The startup model was derived from this same data following 2D Classification and Ab Initio Reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 21879
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.6.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.6.1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 1.6.1)

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