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- EMDB-16859: Structure of BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosome... -

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Basic information

Entry
Database: EMDB / ID: EMD-16859
TitleStructure of BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes (Map1)
Map data
Sample
  • Complex: BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes
    • Complex: DNA, Histones and Polyubiquitin-B
      • DNA: DNA (142-MER)
      • Protein or peptide: Histone H3.1
      • Protein or peptide: Histone H2B type 1-C/E/F/G/I
      • Protein or peptide: Histone H2A type 1
      • Protein or peptide: Histone H4
      • Protein or peptide: Ubiquitin
    • Complex: BRCA1 associated RING domain 1
      • Protein or peptide: BRCA1 associated RING domain 1
KeywordsBRCA1-BARD1 / chromatin recognition / nucleosomes / ubiquitin / DNA BINDING PROTEIN
Function / homology
Function and homology information


BRCA1-BARD1 complex / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / protein K6-linked ubiquitination / regulation of phosphorylation / negative regulation of protein export from nucleus / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes ...BRCA1-BARD1 complex / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / protein K6-linked ubiquitination / regulation of phosphorylation / negative regulation of protein export from nucleus / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / heterochromatin organization / epigenetic regulation of gene expression / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Maturation of protein E / Maturation of protein E / Deposition of new CENPA-containing nucleosomes at the centromere / ER Quality Control Compartment (ERQC) / nucleosomal DNA binding / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Endosomal Sorting Complex Required For Transport (ESCRT) / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / Constitutive Signaling by NOTCH1 HD Domain Mutants / Regulation of FZD by ubiquitination / TICAM1,TRAF6-dependent induction of TAK1 complex / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Inhibition of DNA recombination at telomere / Meiotic synapsis / p75NTR recruits signalling complexes / Downregulation of ERBB4 signaling / APC-Cdc20 mediated degradation of Nek2A / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / PINK1-PRKN Mediated Mitophagy / telomere organization / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Pexophagy / Regulation of innate immune responses to cytosolic DNA / InlA-mediated entry of Listeria monocytogenes into host cells / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / RNA Polymerase I Promoter Opening / NF-kB is activated and signals survival / Interleukin-7 signaling / NRIF signals cell death from the nucleus / Regulation of PTEN localization / Activated NOTCH1 Transmits Signal to the Nucleus / Regulation of BACH1 activity / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Translesion synthesis by REV1 / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLK / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / DNA methylation / Josephin domain DUBs / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Condensation of Prophase Chromosomes / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / innate immune response in mucosa / PRC2 methylates histones and DNA / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / SCF-beta-TrCP mediated degradation of Emi1 / TCF dependent signaling in response to WNT / AUF1 (hnRNP D0) binds and destabilizes mRNA / NIK-->noncanonical NF-kB signaling / Defective pyroptosis / Ubiquitin-dependent degradation of Cyclin D
Similarity search - Function
BARD1, Zinc finger, RING-type / zf-RING of BARD1-type protein / BRCT domain, a BRCA1 C-terminus domain / breast cancer carboxy-terminal domain / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Histone H2B signature. ...BARD1, Zinc finger, RING-type / zf-RING of BARD1-type protein / BRCT domain, a BRCA1 C-terminus domain / breast cancer carboxy-terminal domain / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Ankyrin repeats (3 copies) / Histone H3 signature 1. / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Histone H3 signature 2. / Ankyrin repeat / Histone H3 / Histone H3/CENP-A / Zinc finger RING-type profile. / Zinc finger, RING-type / Ankyrin repeat-containing domain superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Histone-fold / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
BRCA1 associated RING domain 1 / Histone H2A type 1 / Polyubiquitin-C / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.1
Similarity search - Component
Biological speciesHomo sapiens (human) / Felis catus (domestic cat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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
DepositionMar 15, 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_16859.png

Downloads & links

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Map

FileDownload / File: emd_16859.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.435
Minimum - Maximum-2.1360242 - 3.2577093
Average (Standard dev.)0.008429022 (±0.081438005)
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_16859_msk_1.map
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Additional map: #1

Fileemd_16859_additional_1.map
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Additional map: #2

Fileemd_16859_additional_2.map
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Half map: #2

Fileemd_16859_half_map_1.map
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Half map: #1

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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
      • DNA: DNA (142-MER)
      • Protein or peptide: Histone H3.1
      • Protein or peptide: Histone H2B type 1-C/E/F/G/I
      • Protein or peptide: Histone H2A type 1
      • Protein or peptide: Histone H4
      • Protein or peptide: Ubiquitin
    • Complex: BRCA1 associated RING domain 1
      • Protein or peptide: 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: all
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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Macromolecule #1: DNA (142-MER)

MacromoleculeName: DNA (142-MER) / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 108.097586 KDa
SequenceString: (DA)(DA)(DT)(DA)(DC)(DG)(DC)(DG)(DG)(DC) (DC)(DG)(DC)(DC)(DC)(DT)(DG)(DG)(DA)(DG) (DA)(DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT) (DG)(DC)(DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG) (DC) (DT)(DC)(DA)(DA)(DT)(DT) ...String:
(DA)(DA)(DT)(DA)(DC)(DG)(DC)(DG)(DG)(DC) (DC)(DG)(DC)(DC)(DC)(DT)(DG)(DG)(DA)(DG) (DA)(DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT) (DG)(DC)(DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG) (DC) (DT)(DC)(DA)(DA)(DT)(DT)(DG)(DG) (DT)(DC)(DG)(DT)(DA)(DG)(DA)(DC)(DA)(DG) (DC)(DT) (DC)(DT)(DA)(DG)(DC)(DA)(DC) (DC)(DG)(DC)(DT)(DT)(DA)(DA)(DA)(DC)(DG) (DC)(DA)(DC) (DG)(DT)(DA)(DC)(DG)(DC) (DG)(DC)(DT)(DG)(DT)(DC)(DC)(DG)(DC)(DC) (DG)(DC)(DG)(DT) (DT)(DT)(DT)(DA)(DA) (DC)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DG)(DG) (DG)(DA)(DT)(DT)(DA) (DC)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC)(DC)(DA) (DG)(DG)(DC)(DA)(DC)(DG) (DT)(DG)(DT) (DC)(DA)(DG)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DC)(DA)(DT)(DC)(DC)(DT)(DG) (DT)(DG) (DC)(DA)(DA)(DT)(DG)(DT)(DG)(DT)(DT)(DC) (DC)(DA)(DT)(DA)(DT)(DG)(DG)(DA) (DA) (DC)(DA)(DC)(DA)(DT)(DT)(DG)(DC)(DA)(DC) (DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DA) (DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG)(DA) (DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG)(DT)(DA)(DA)(DT)(DC)(DC)(DC)(DC)(DT) (DT) (DG)(DG)(DC)(DG)(DG)(DT)(DT)(DA) (DA)(DA)(DA)(DC)(DG)(DC)(DG)(DG)(DC)(DG) (DG)(DA) (DC)(DA)(DG)(DC)(DG)(DC)(DG) (DT)(DA)(DC)(DG)(DT)(DG)(DC)(DG)(DT)(DT) (DT)(DA)(DA) (DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG)(DA)(DG)(DC)(DT)(DG)(DT) (DC)(DT)(DA)(DC) (DG)(DA)(DC)(DC)(DA) (DA)(DT)(DT)(DG)(DA)(DG)(DC)(DG)(DG)(DC) (DC)(DT)(DC)(DG)(DG) (DC)(DA)(DC)(DC) (DG)(DG)(DG)(DA)(DT)(DT)(DC)(DT)(DC)(DC) (DA)(DG)(DG)(DG)(DC)(DG) (DG)(DC)(DC) (DG)(DC)(DG)(DT)(DA)(DT)(DT)

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Macromolecule #2: Histone H3.1

MacromoleculeName: Histone H3.1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.366088 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSSA VMALQEACEA YLVGLFEDTN LCAIHAKRVT IMPKDIQLAR RIRGER

UniProtKB: Histone H3.1

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Macromolecule #3: Histone H2B type 1-C/E/F/G/I

MacromoleculeName: Histone H2B type 1-C/E/F/G/I / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.937213 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSV YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSSK

UniProtKB: Histone H2B type 1-C/E/F/G/I

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Macromolecule #4: Histone H2A type 1

MacromoleculeName: Histone H2A type 1 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.121537 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK ARAKAKTRSS RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIR NDEELNKLLG KVTIAQGGVL PNIQAVLLPK KTESHHKAKG K

UniProtKB: Histone H2A type 1

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Macromolecule #5: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.394426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVY ALKRQGRTLY GFGG

UniProtKB: Histone H4

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Macromolecule #6: BRCA1 associated RING domain 1

MacromoleculeName: BRCA1 associated RING domain 1 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Felis catus (domestic cat)
Molecular weightTheoretical: 85.897531 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPGNRQLRVR SGNEPRPAPA MEPEGRGAWA HSRAAFDRLE KLLRCSRCTN ILREPVCLGG CEHIFCSNCV SDCIGTGCPV CYTPAWIQD VKINRQLDSM IQLCSKLRNL LHDNKLSDLK EDTSRQNVFN DAENKKNSIK MWFSPRSKKV RYVVTKVSVQ T QPQVIDDG ...String:
MPGNRQLRVR SGNEPRPAPA MEPEGRGAWA HSRAAFDRLE KLLRCSRCTN ILREPVCLGG CEHIFCSNCV SDCIGTGCPV CYTPAWIQD VKINRQLDSM IQLCSKLRNL LHDNKLSDLK EDTSRQNVFN DAENKKNSIK MWFSPRSKKV RYVVTKVSVQ T QPQVIDDG NAQQASVYKF VSTSPPTSVP ERAKKASTRS RKKQKKKTLA EINQEWNFEA EKEDGEPDSK EEFKEKLVSF CS QPSVIAS PQTNGGIDLL ASDSVTESEC SRSLTEVSLP LAEQIESPET GSRNEDATPE KNACVDHLTS KQPLPSGHNG RPG RRSRRS SPVSKRCRSS IPGTSGQQML LSENKPLPGC SSPPPSKRKV GDTLRRKNSN ISDESMSLSP GTPPSTLNSP SYRR MMFSP SAVKLSPGSL TAVKRNHRGE TLLHIASIKG DLPSVEYLLQ NGSDPNVKDH AGWTPLHEAC NHGHLKVVEL LLQHK ALVN STGYQNDSPL HDAAKNGHMD IVKLLLSYGA SRNAVNIFGL RPVDYADGEN MKSLLLLPEQ NESSSTRHCS VTNTGQ RRD GPLVLIGSGL SSEQQKMLSE LAVILKAKKC AEFDSTVTHV IVPGDTVQST LKCMLGILNG CWILKFEWVK ACLQRKA CE QEEKYEIPEG PQRSRLNKEQ LLPKLFDGCY FYFGGAFKHH PKDNLVKLVT AAGGQVLSRK PKPDSDVTQT INTVAYHA K ADSDQRFCTQ YIIYEDLSNH RPERVRQGKV WMAPSSWFID CVMSFELLPL DS

UniProtKB: BRCA1 associated RING domain 1

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Macromolecule #7: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.576831 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

UniProtKB: Polyubiquitin-C

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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: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 359954
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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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8off:
Structure of BARD1 ARD-BRCTs in complex with H2AKc15ub nucleosomes (Map1)

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