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Entry
Database: PDB / ID: 8sn6
TitleCryo-EM structure of the human nucleosome core particle in complex with RNF168 and UbcH5c~Ub (UbcH5c chemically conjugated to histone H2A) (class 4)
Components
  • (DNA (147-MER)) x 2
  • E3 ubiquitin-protein ligase RNF168
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1Histone H3
  • Histone H4
  • Polyubiquitin-B
  • Ubiquitin-conjugating enzyme E2 D3
KeywordsTRANSFERASE/DNA / Nucleosome core particle / chromatin / RNF168 / RING domain / UbcH5c / DNA repair / DNA double-strand break / Homologous recombination / 53BP1 / ubiquitin / STRUCTURAL PROTEIN-DNA-TRANSFERASE complex / TRANSFERASE / TRANSFERASE-DNA complex
Function / homology
Function and homology information


histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / (E3-independent) E2 ubiquitin-conjugating enzyme / Signaling by BMP / protein K6-linked ubiquitination / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I ...histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / (E3-independent) E2 ubiquitin-conjugating enzyme / Signaling by BMP / protein K6-linked ubiquitination / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / DNA repair-dependent chromatin remodeling / positive regulation of protein monoubiquitination / mitochondrion transport along microtubule / positive regulation of protein targeting to mitochondrion / fat pad development / K63-linked polyubiquitin modification-dependent protein binding / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / female gonad development / seminiferous tubule development / male meiosis I / negative regulation of transcription elongation by RNA polymerase II / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / protein monoubiquitination / ubiquitin conjugating enzyme activity / protein K63-linked ubiquitination / negative regulation of BMP signaling pathway / heterochromatin organization / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / nucleosomal DNA binding / protein localization to CENP-A containing chromatin / protein autoubiquitination / protein K48-linked ubiquitination / nucleosome binding / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / interstrand cross-link repair / CENP-A containing nucleosome / SUMOylation of DNA damage response and repair proteins / epigenetic regulation of gene expression / energy homeostasis / regulation of neuron apoptotic process / Packaging Of Telomere Ends / regulation of proteasomal protein catabolic process / ubiquitin ligase complex / 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) / 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 / positive regulation of DNA repair / Downregulation of ERBB4 signaling / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / telomere organization / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / InlA-mediated entry of Listeria monocytogenes into host cells / 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
Similarity search - Function
E3 ubiquitin-protein ligase RNF168 / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like ...E3 ubiquitin-protein ligase RNF168 / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like / Ring finger / 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 / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Zinc finger RING-type profile. / Zinc finger, RING-type / 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
DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1-B/E / Histone H2B type 1-J / Polyubiquitin-B / Ubiquitin-conjugating enzyme E2 D3 / Histone H4 / Histone H3.1 / E3 ubiquitin-protein ligase RNF168
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsHu, Q. / Botuyan, M.V. / Zhao, D. / Cui, G. / Mer, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM136262 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA132878 United States
CitationJournal: Mol Cell / Year: 2024
Title: Mechanisms of RNF168 nucleosome recognition and ubiquitylation.
Authors: Qi Hu / Debiao Zhao / Gaofeng Cui / Janarjan Bhandari / James R Thompson / Maria Victoria Botuyan / Georges Mer /
Abstract: RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for ...RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.
History
DepositionApr 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 20, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.1
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.1
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (147-MER)
J: DNA (147-MER)
K: E3 ubiquitin-protein ligase RNF168
L: Ubiquitin-conjugating enzyme E2 D3
M: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,14815
Polymers238,01713
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 7 types, 11 molecules AEBFCGDHKLM

#1: Protein Histone H3.1 / Histone H3 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone ...Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone H3/i / Histone H3/j / Histone H3/k / Histone H3/l


Mass: 15786.534 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H3C1, H3FA, HIST1H3A, H3C2, H3FL, HIST1H3B, H3C3, H3FC HIST1H3C, H3C4, H3FB, HIST1H3D, H3C6, H3FD, HIST1H3E, H3C7, H3FI, HIST1H3F, H3C8, H3FH, HIST1H3G, H3C10, H3FK, HIST1H3H, H3C11, H3FF, ...Gene: H3C1, H3FA, HIST1H3A, H3C2, H3FL, HIST1H3B, H3C3, H3FC HIST1H3C, H3C4, H3FB, HIST1H3D, H3C6, H3FD, HIST1H3E, H3C7, H3FI, HIST1H3F, H3C8, H3FH, HIST1H3G, H3C10, H3FK, HIST1H3H, H3C11, H3FF, HIST1H3I, H3C12, H3FJ, HIST1H3J
Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P68431
#2: Protein Histone H4 /


Mass: 11743.792 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
Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 13008.156 Da / Num. of mol.: 2 / Mutation: R11S, K15C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 14084.348 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC11, H2BFR, HIST1H2BJ / Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P06899
#7: Protein E3 ubiquitin-protein ligase RNF168 / hRNF168 / RING finger protein 168 / RING-type E3 ubiquitin transferase RNF168


Mass: 11903.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF168 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8IYW5, RING-type E3 ubiquitin transferase
#8: Protein Ubiquitin-conjugating enzyme E2 D3 / (E3-independent) E2 ubiquitin-conjugating enzyme D3 / E2 ubiquitin-conjugating enzyme D3 / ...(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: 17061.418 Da / Num. of mol.: 1 / Mutation: C21I, C107A, C111D, L119K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2D3, UBC5C, UBCH5C / Production host: Escherichia coli (E. coli)
References: UniProt: P61077, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme
#9: Protein Polyubiquitin-B


Mass: 9057.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG47

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (147-MER)


Mass: 45610.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 1 types, 2 molecules

#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: Human nucleosome core particle in complex with RNF168 and UbcH5c~UbNucleosome
Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.2565 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 10 mM HEPES, 100 mM NaCl, 1 mM DTT, pH 7.5
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14179
Details: 14179 images were recorded in movie-mode of which 14113 were retained for particle picking.

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.12particle selection
2EPUimage acquisition
4cryoSPARC4.12CTF correction
7PHENIX1.18.2-3874model fitting
9PHENIX1.18.2-3874model refinement
10cryoSPARC4.12initial Euler assignment
11cryoSPARC4.12final Euler assignment
12cryoSPARC4.12classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 16063830
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33609 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17LYA

7lya

17LYA1PDBexperimental model
24GB0

4gb0

14GB02PDBexperimental model
35EGG

5egg

15EGG3PDBexperimental model
41UBQ

1ubq

11UBQ4PDBexperimental model

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