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- PDB-7jzv: Cryo-EM structure of the BRCA1-UbcH5c/BARD1 E3-E2 module bound to... -

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Entry
Database: PDB / ID: 7jzv
TitleCryo-EM structure of the BRCA1-UbcH5c/BARD1 E3-E2 module bound to a nucleosome
Components
  • (Widom 601 153- ...) x 2
  • BRCA1,Ubiquitin-conjugating enzyme E2 D3
  • BRCA1-associated RING domain protein 1
  • Histone H2A type 2-A
  • Histone H2B type 1-K
  • Histone H3.2
  • Histone H4
KeywordsLIGASE/DNA / Complex / Ubiquitin / Nucleosome / RING / ligase / LIGASE-DNA complex
Function / homology
Function and homology information


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-B complex / BRCA1-A complex / BRCA1-C complex / negative regulation of centriole replication ...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-B complex / BRCA1-A complex / BRCA1-C complex / negative regulation of centriole replication / sex-chromosome dosage compensation / random inactivation of X chromosome / ubiquitin-modified histone reader activity / chordate embryonic development / cellular response to indole-3-methanol / gamma-tubulin ring complex / negative regulation of intracellular estrogen receptor signaling pathway / nuclear ubiquitin ligase complex / DNA strand resection involved in replication fork processing / negative regulation of fatty acid biosynthetic process / homologous recombination / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / tissue homeostasis / Signaling by BMP / protein K6-linked ubiquitination / regulation of phosphorylation / (E3-independent) E2 ubiquitin-conjugating enzyme / lateral element / Impaired BRCA2 binding to PALB2 / regulation of DNA damage checkpoint / XY body / mitotic G2/M transition checkpoint / negative regulation of protein export from nucleus / protein K11-linked ubiquitination / RNA polymerase binding / DNA repair complex / DNA damage tolerance / centrosome cycle / 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 / E2 ubiquitin-conjugating enzyme / intracellular membraneless organelle / HDR through Single Strand Annealing (SSA) / response to ionizing radiation / negative regulation of gene expression via chromosomal CpG island methylation / Impaired BRCA2 binding to RAD51 / Transcriptional Regulation by E2F6 / mitotic G2 DNA damage checkpoint signaling / negative regulation of cell cycle / ubiquitin conjugating enzyme activity / 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 / ubiquitin ligase complex / SUMOylation of DNA damage response and repair proteins / protein K48-linked ubiquitination / regulation of DNA repair / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / protein autoubiquitination / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / tubulin binding / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / Inhibition of DNA recombination at telomere / Assembly of the ORC complex at the origin of replication / Meiotic synapsis / positive regulation of DNA repair / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / TICAM1, RIP1-mediated IKK complex recruitment / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / IKK complex recruitment mediated by RIP1 / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PINK1-PRKN Mediated Mitophagy / PRC2 methylates histones and DNA / innate immune response in mucosa / cellular response to ionizing radiation / Regulation of endogenous retroelements by KRAB-ZFP proteins
Similarity search - Function
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 / Ubiquitin Conjugating Enzyme / Ubiquitin Conjugating Enzyme / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) ...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 / Ubiquitin Conjugating Enzyme / Ubiquitin Conjugating Enzyme / 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. / Zinc/RING finger domain, C3HC4 (zinc finger) / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Herpes Virus-1 / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / breast cancer carboxy-terminal domain / Ubiquitin-conjugating enzyme/RWD-like / 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 / Zinc finger, RING-type / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold / Zinc finger, RING/FYVE/PHD-type / Roll / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Truncated breast and ovarian cancer susceptibility protein 1 / Histone H2B type 1-K / Breast cancer type 1 susceptibility protein / Ubiquitin-conjugating enzyme E2 D3 / Histone H4 / Histone H2A type 2-A / Histone H3.2 / BRCA1-associated RING domain protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsWitus, S.R. / Burrell, A.L. / Hansen, J.M. / Farrell, D.P. / Dimaio, F. / Kollman, J.M. / Klevit, R.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088055 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.
Authors: Samuel R Witus / Anika L Burrell / Daniel P Farrell / Jianming Kang / Meiling Wang / Jesse M Hansen / Alex Pravat / Lisa M Tuttle / Mikaela D Stewart / Peter S Brzovic / Champak Chatterjee / ...Authors: Samuel R Witus / Anika L Burrell / Daniel P Farrell / Jianming Kang / Meiling Wang / Jesse M Hansen / Alex Pravat / Lisa M Tuttle / Mikaela D Stewart / Peter S Brzovic / Champak Chatterjee / Weixing Zhao / Frank DiMaio / Justin M Kollman / Rachel E Klevit /
Abstract: Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme ...Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
History
DepositionSep 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: BRCA1,Ubiquitin-conjugating enzyme E2 D3
B: BRCA1-associated RING domain protein 1
N: Histone H2A type 2-A
O: Histone H2B type 1-K
P: Histone H3.2
Q: Histone H4
X: Widom 601 153-bp
Y: Widom 601 153-bp
n: Histone H2A type 2-A
o: Histone H2B type 1-K
p: Histone H3.2
q: Histone H4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,91916
Polymers245,65712
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 6 types, 10 molecules ABNnOoPpQq

#1: Protein BRCA1,Ubiquitin-conjugating enzyme E2 D3 / BRCA1-UbcH5c / (E3-independent) E2 ubiquitin-conjugating enzyme D3 / E2 ubiquitin-conjugating ...BRCA1-UbcH5c / (E3-independent) E2 ubiquitin-conjugating enzyme D3 / E2 ubiquitin-conjugating enzyme D3 / Ubiquitin carrier protein D3 / Ubiquitin-conjugating enzyme E2(17)KB 3 / Ubiquitin-conjugating enzyme E2-17 kDa 3 / Ubiquitin-protein ligase D3


Mass: 28974.465 Da / Num. of mol.: 1 / Mutation: UbcH5c C85K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRCA1, UBE2D3, UBC5C, UBCH5C / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A386IN42, UniProt: P61077, UniProt: P38398*PLUS, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme, RING-type E3 ubiquitin transferase
#2: Protein BRCA1-associated RING domain protein 1 / BARD-1 / RING-type E3 ubiquitin transferase BARD1


Mass: 12983.018 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BARD1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q99728, RING-type E3 ubiquitin transferase
#3: Protein Histone H2A type 2-A / H2A-clustered histone 18 / H2A-clustered histone 19 / Histone H2A.2 / Histone H2A/o


Mass: 14310.728 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H2AC18, H2AFO, HIST2H2AA, HIST2H2AA3, H2AC19, HIST2H2AA4
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6FI13
#4: Protein Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 13790.018 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC12, H2BFT, HIRIP1, HIST1H2BK / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O60814
#5: Protein Histone H3.2 / H3-clustered histone 13 / H3-clustered histone 14 / H3-clustered histone 15 / Histone H3/m / Histone H3/o


Mass: 15257.838 Da / Num. of mol.: 2 / Mutation: C110A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H3C15, HIST2H3A, H3C14, H3F2, H3FM, HIST2H3C, H3C13, HIST2H3D
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q71DI3
#6: Protein Histone H4


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H4C1, H4/A, H4FA, HIST1H4A, H4C2, H4/I, H4FI, HIST1H4B, H4C3, H4/G, H4FG, HIST1H4C, H4C4, H4/B, H4FB, HIST1H4D, H4C5, H4/J, H4FJ, HIST1H4E, H4C6, H4/C, H4FC, HIST1H4F, H4C8, H4/H, H4FH, ...Gene: H4C1, H4/A, H4FA, HIST1H4A, H4C2, H4/I, H4FI, HIST1H4B, H4C3, H4/G, H4FG, HIST1H4C, H4C4, H4/B, H4FB, HIST1H4D, H4C5, H4/J, H4FJ, HIST1H4E, H4C6, H4/C, H4FC, HIST1H4F, H4C8, H4/H, H4FH, HIST1H4H, H4C9, H4/M, H4FM, HIST1H4I, H4C11, H4/E, H4FE, HIST1H4J, H4C12, H4/D, H4FD, HIST1H4K, H4C13, H4/K, H4FK, HIST1H4L, H4C14, H4/N, H4F2, H4FN, HIST2H4, HIST2H4A, H4C15, H4/O, H4FO, HIST2H4B, H4-16, HIST4H4
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62805

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Widom 601 153- ... , 2 types, 2 molecules XY

#7: DNA chain Widom 601 153-bp


Mass: 47457.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): DH5a
#8: DNA chain Widom 601 153-bp


Mass: 46998.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): DH5a

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Non-polymers , 1 types, 4 molecules

#9: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1BRCA1-UbcH5c/BARD1 E3-E2 module bound to nucleosome core particleCOMPLEX#1-#80RECOMBINANT
2BRCA1-UbcH5c/BARD1 E3-E2 moduleCOMPLEX#1-#21RECOMBINANT
3Nucleosome core particleCOMPLEX#3-#81RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.24 MDaNO
210.04 MDaNO
310.2 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21synthetic construct (others)32630
32Homo sapiens (human)9606
43Homo sapiens (human)9606
53synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli BL21(DE3) (bacteria)469008
22Escherichia coli BL21(DE3) (bacteria)469008
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES-NaOH1
235 mMNaCl1
31 mMDTT1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 90 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1cisTEMparticle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
19RosettaEMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21479 / Symmetry type: POINT

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