EMDB-23591: Cryo-EM structure of the human nucleosome core particle in comple...

Basic information

• Entry: Database: EMDB / ID: EMD-23591
• Title: Cryo-EM structure of the human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c

Sample

• Complex: Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c
  • Protein or peptide: Histone H3.1
  • Protein or peptide: Histone H4
  • Protein or peptide: Histone H2B type 1-J
  • DNA: DNA (147-MER)
  • DNA: DNA (146-MER)
  • Protein or peptide: Isoform 7 of Breast cancer type 1 susceptibility protein
  • Protein or peptide: Ubiquitin-conjugating enzyme E2 D3
  • Protein or peptide: BRCA1-associated RING domain protein 1
  • Protein or peptide: Histone H2A type 1-B/E
• Ligand: ZINC ION

• Keywords: Nucleosome core particle / chromatin / BRCA1 / BARD1 / UbcH5c / DNA repair / DNA double-strand break / Homologous recombination / 53BP1 / STRUCTURAL PROTEIN-DNA complex

Function / homology

Function:

negative regulation of mRNA 3'-end processing / histone H2AK127 ubiquitin ligase activity / histone H2AK129 ubiquitin ligase activity / Defective DNA double strand break response due to BRCA1 loss of function / Defective DNA double strand break response due to BARD1 loss of function / BRCA1-BARD1 complex / BRCA1-C complex / BRCA1-B complex / BRCA1-A complex / negative regulation of centriole replication / sex-chromosome dosage compensation / random inactivation of X chromosome / nuclear ubiquitin ligase complex / ubiquitin-modified histone reader activity / chordate embryonic development / negative regulation of intracellular estrogen receptor signaling pathway / cellular response to indole-3-methanol / gamma-tubulin ring complex / negative regulation of fatty acid biosynthetic process / DNA strand resection involved in replication fork processing / homologous recombination / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / tissue homeostasis / protein K6-linked ubiquitination / regulation of phosphorylation / Signaling by BMP / (E3-independent) E2 ubiquitin-conjugating enzyme / lateral element / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / XY body / mitotic G2/M transition checkpoint / negative regulation of protein export from nucleus / protein K11-linked ubiquitination / centrosome cycle / RNA polymerase binding / postreplication repair / DNA repair complex / positive regulation of protein targeting to mitochondrion / Homologous DNA Pairing and Strand Exchange / 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 / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / intracellular membraneless organelle / HDR through Single Strand Annealing (SSA) / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / negative regulation of gene expression via chromosomal CpG island methylation / Impaired BRCA2 binding to RAD51 / mitotic G2 DNA damage checkpoint signaling / Transcriptional Regulation by E2F6 / ubiquitin conjugating enzyme activity / negative regulation of cell cycle / negative regulation of reactive oxygen species metabolic process / Presynaptic phase of homologous DNA pairing and strand exchange / positive regulation of vascular endothelial growth factor production / negative regulation of BMP signaling pathway / protein monoubiquitination / negative regulation of tumor necrosis factor-mediated signaling pathway / ubiquitin ligase complex / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / SUMOylation of DNA damage response and repair proteins / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / protein K48-linked ubiquitination / regulation of DNA repair / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / protein autoubiquitination / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / TICAM1, RIP1-mediated IKK complex recruitment / IKK complex recruitment mediated by RIP1 / Deposition of new CENPA-containing nucleosomes at the centromere / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / PINK1-PRKN Mediated Mitophagy / telomere organization / Interleukin-7 signaling / Inhibition of DNA recombination at telomere / RNA Polymerase I Promoter Opening / Meiotic synapsis / cellular response to ionizing radiation / tubulin binding / Assembly of the ORC complex at the origin of replication / Negative regulators of DDX58/IFIH1 signaling / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / positive regulation of DNA repair / DNA methylation / Condensation of Prophase Chromosomes / Peroxisomal protein import / Chromatin modifications during the maternal to zygotic transition (MZT) / male germ cell nucleus / epigenetic regulation of gene expression / HCMV Late Events

Domain/homology:

BARD1, Zinc finger, RING-type / zf-RING of BARD1-type protein / Breast cancer type 1 susceptibility protein (BRCA1) / BRCA1, serine-rich domain / BRCA1-associated / Serine-rich domain associated with BRCT / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / BRCA1 C Terminus (BRCT) domain / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Zinc finger RING-type profile. / Ankyrin repeat-containing domain superfamily / Histone H3 signature 1. / Zinc finger, RING-type / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Zinc finger, RING/FYVE/PHD-type

Component:

Histone H2A type 1-B/E / Histone H2B type 1-J / Breast cancer type 1 susceptibility protein / Ubiquitin-conjugating enzyme E2 D3 / Histone H4 / Histone H3.1 / BRCA1-associated RING domain protein 1

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.28 Å
• Authors: Hu Q / Botuyan MV

Funding support

United States, 3 items

Organization	Grant number	Country
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01 CA132878	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	R35 GM136262	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	R01 GM116829	United States

• Citation: Journal: Nature / Year: 2021; Title: Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.; Authors: Qi Hu / Maria Victoria Botuyan / Debiao Zhao / Gaofeng Cui / Elie Mer / Georges Mer / ; Abstract: The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks. We further show that RING domains in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

History

Deposition	Mar 6, 2021	-
Header (metadata) release	Jul 28, 2021	-
Map release	Jul 28, 2021	-
Update	Oct 23, 2024	-
Current status	Oct 23, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_23591.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.031 Å

Density

Contour Level	By AUTHOR: 0.08 / Movie #1: 0.1
Minimum - Maximum	-0.27242526 - 0.6002898
Average (Standard dev.)	0.00006332425 (±0.022572892)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 263.936 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.031	1.031	1.031
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	263.936	263.936	263.936
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	300	300	300
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	256	256	256
D min/max/mean	-0.272	0.600	0.000

Supplemental data

Mask #1

• File: emd_23591_msk_1.map

Additional map: #1

• File: emd_23591_additional_1.map

Half map: #2

• File: emd_23591_half_map_1.map

Half map: #1

• File: emd_23591_half_map_2.map

Sample components

Entire : Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c

• Entire: Name: Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c

Components

• Complex: Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c
  • Protein or peptide: Histone H3.1
  • Protein or peptide: Histone H4
  • Protein or peptide: Histone H2B type 1-J
  • DNA: DNA (147-MER)
  • DNA: DNA (146-MER)
  • Protein or peptide: Isoform 7 of Breast cancer type 1 susceptibility protein
  • Protein or peptide: Ubiquitin-conjugating enzyme E2 D3
  • Protein or peptide: BRCA1-associated RING domain protein 1
  • Protein or peptide: Histone H2A type 1-B/E
• Ligand: ZINC ION

Supramolecule #1: Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c

• Supramolecule: Name: Human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Histone H3.1

• Macromolecule: Name: Histone H3.1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 15.786534 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GPGHMARTKQ TARKSTGGKA PRKQLATKAA RKSAPATGGV KKPHRYRPGT VALREIRRYQ KSTELLIRKL PFQRLVREIA QDFKTDLRF QSSAVMALQE ACEAYLVGLF EDTNLCAIHA KRVTIMPKDI QLARRIRGER A

UniProtKB: Histone H3.1

Macromolecule #2: Histone H4

• Macromolecule: Name: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 11.743792 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GPGHMSGRGK GGKGLGKGGA KRHRKVLRDN IQGITKPAIR RLARRGGVKR ISGLIYEETR GVLKVFLENV IRDAVTYTEH AKRKTVTAM DVVYALKRQG RTLYGFGG

UniProtKB: Histone H4

Macromolecule #3: Histone H2B type 1-J

• Macromolecule: Name: Histone H2B type 1-J / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 13.930181 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GHMPEPAKSA PAPKKGSKKA VTKAQKKDGK KRKRSRKESY SIYVYKVLKQ VHPDTGISSK AMGIMNSFVN DIFERIAGEA SRLAHYNKR STITSREIQT AVRLLLPGEL AKHAVSEGTK AVTKYTS

UniProtKB: Histone H2B type 1-J

Macromolecule #6: Isoform 7 of Breast cancer type 1 susceptibility protein

• Macromolecule: Name: Isoform 7 of Breast cancer type 1 susceptibility protein; type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 13.053065 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GGSGGSGGSG GSGGSGGSGG SGGSMDLSAL RVEEVQNVIN AMQKILECPI CLELIKEPVS TKCDHIFCKF CMLKLLNQKK GPSQCPLCK NDITKRSLQE STRFSQLVEE LLKIICAFQL DTGLE

UniProtKB: Breast cancer type 1 susceptibility protein

Macromolecule #7: Ubiquitin-conjugating enzyme E2 D3

• Macromolecule: Name: Ubiquitin-conjugating enzyme E2 D3 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 17.869113 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GGSGGSGGSG GSGGSGGSSA LKRINKELSD LARDPPAQCS AGPVGDDMFH WQATIMGPND SPYQGGVFFL TIHFPTDYPF KPPKVAFTT RIYHPNINSN GSICLDILRS QWSPALTISK VLLSICSLLC DPNPDDPLVP EIARIYKTDR DKYNRISREW T QKYAM

UniProtKB: Ubiquitin-conjugating enzyme E2 D3

Macromolecule #8: BRCA1-associated RING domain protein 1

• Macromolecule: Name: BRCA1-associated RING domain protein 1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 10.906617 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MEPDGRGAWA HSRAALDRLE KLLRCSRCTN ILREPVCLGG CEHIFCSNCV SDCIGTGCPV CYTPAWIQDL KINRQLDSMI QLCSKLRNL LHDNELSD

UniProtKB: BRCA1-associated RING domain protein 1

Macromolecule #9: Histone H2A type 1-B/E

• Macromolecule: Name: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 9 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 13.034193 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

SAKAKTRSSR AGLQFPVGRV HRLLRKGNYS ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGR VTIAQGGVLP NIQAVLLPKK TESHHKAKGK

UniProtKB: Histone H2A type 1-B/E

Macromolecule #4: DNA (147-MER)

• Macromolecule: Name: DNA (147-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 45.13877 KDa

Sequence

String:

(DA)(DT)(DC)(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)(DC) (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)(DG)(DA)(DT)

Macromolecule #5: DNA (146-MER)

• Macromolecule: Name: DNA (146-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 45.610043 KDa

Sequence

String:

(DA)(DT)(DC)(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) (DG)(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)(DG)(DA)(DT)

Macromolecule #10: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 10 / Number of copies: 4 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.3 mg/mL
• Buffer: pH: 7.5
• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: 10 mM HEPES, 100 mM NaCl, 1 mM DTT, pH 7.5

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 5490 / Average electron dose: 1.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 6380972
• Startup model: Type of model: NONE
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 74575
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14)
• Final 3D classification: Software - Name: cryoSPARC (ver. 2.14)

Atomic model buiding 1

Initial model

PDB ID	Chain
1jm7	source_name: PDB, initial_model_type: experimental model
5egg	source_name: PDB, initial_model_type: experimental model

• Refinement: Space: REAL / Protocol: RIGID BODY FIT

Output model

PDB-7lyb:
Cryo-EM structure of the human nucleosome core particle in complex with BRCA1-BARD1-UbcH5c