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Entry
Database: PDB / ID: 8upf
TitleCryo-EM structure of the human nucleosome core particle in complex with RNF168-UbcH5c
Components
  • (DNA (147-MER)) x 2
  • E3 ubiquitin-protein ligase RNF168
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1
  • Histone H4
  • Ubiquitin-conjugating enzyme E2 D3
KeywordsTRANSFERASE/DNA / Nucleosome core particle / chromatin / RNF168 / RING domain / UbcH5c / DNA repair / DNA double-strand break / Homologous recombination / 53BP1 / BRCA1-BARD1 / 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 / Signaling by BMP / protein K6-linked ubiquitination / (E3-independent) E2 ubiquitin-conjugating enzyme / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / K63-linked polyubiquitin modification-dependent protein binding ...histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / Signaling by BMP / protein K6-linked ubiquitination / (E3-independent) E2 ubiquitin-conjugating enzyme / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / K63-linked polyubiquitin modification-dependent protein binding / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / DNA repair-dependent chromatin remodeling / ubiquitin conjugating enzyme activity / negative regulation of transcription elongation by RNA polymerase II / protein K63-linked ubiquitination / protein monoubiquitination / negative regulation of BMP signaling pathway / ubiquitin ligase complex / negative regulation of tumor necrosis factor-mediated signaling pathway / interstrand cross-link repair / SUMOylation of DNA damage response and repair proteins / protein K48-linked ubiquitination / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / protein autoubiquitination / nucleosome binding / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / telomere organization / Interleukin-7 signaling / Inhibition of DNA recombination at telomere / RNA Polymerase I Promoter Opening / Meiotic synapsis / Assembly of the ORC complex at the origin of replication / positive regulation of DNA repair / TICAM1, RIP1-mediated IKK complex recruitment / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / epigenetic regulation of gene expression / Condensation of Prophase Chromosomes / IKK complex recruitment mediated by RIP1 / ubiquitin binding / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / PINK1-PRKN Mediated Mitophagy / innate immune response in mucosa / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Negative regulators of DDX58/IFIH1 signaling / HDACs deacetylate histones / Peroxisomal protein import / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / Regulation of TNFR1 signaling / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / HDMs demethylate histones / Inactivation of CSF3 (G-CSF) signaling / G2/M DNA damage checkpoint / RING-type E3 ubiquitin transferase / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / double-strand break repair via nonhomologous end joining / protein modification process / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / protein polyubiquitination / HCMV Early Events / ubiquitin-protein transferase activity / antimicrobial humoral immune response mediated by antimicrobial peptide / ubiquitin protein ligase activity / structural constituent of chromatin / UCH proteinases / antibacterial humoral response / Antigen processing: Ubiquitination & Proteasome degradation / nucleosome
Similarity search - Function
E3 ubiquitin-protein ligase RNF168 / : / Prokaryotic RING finger family 4 / 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 ...E3 ubiquitin-protein ligase RNF168 / : / Prokaryotic RING finger family 4 / 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 / 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 / Zinc finger RING-type profile. / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1-B/E / Histone H2B type 1-J / 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: 3.2 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
DepositionOct 22, 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
Revision 1.3Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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
D: Histone H2B type 1-J
E: Histone H3.1
F: Histone H4
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
C: Histone H2A type 1-B/E
G: Histone H2A type 1-B/E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,30014
Polymers228,17012
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 6 types, 10 molecules AEBFDHKLCG

#1: Protein Histone H3.1 / 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 H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 13209.365 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
#6: Protein E3 ubiquitin-protein ligase RNF168 / hRNF168 / RING finger protein 168 / RING-type E3 ubiquitin transferase RNF168


Mass: 11370.299 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
#7: 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: 18502.672 Da / Num. of mol.: 1
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
#8: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 13034.193 Da / Num. of mol.: 2 / Mutation: R11S
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

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DNA chain , 2 types, 2 molecules IJ

#4: DNA chain DNA (147-MER)


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


Mass: 45610.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 1 types, 2 molecules

#9: 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
Has protein modificationY

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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-UbcH5c
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.2565 MDa / Experimental value: YES
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 mesh size: 300 divisions/in. / 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 FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4867
Details: 4867 images were recorded in movie-mode of which 4644 were retained for particle picking.

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC2.14CTF correction
7PHENIX1.18.2-3874model fitting
9cryoSPARC2.14initial Euler assignment
10cryoSPARC2.14final Euler assignment
11cryoSPARC2.14classification
12cryoSPARC2.143D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7889111
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58264 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDSource nameDetails
14GB0

4gb0

4GB01PDB
25EGG

5egg

5EGGPDB
3OtherUnpublished crystal structure of the human nucleosome core particle.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00714877
ELECTRON MICROSCOPYf_angle_d0.63321366
ELECTRON MICROSCOPYf_dihedral_angle_d21.4688534
ELECTRON MICROSCOPYf_chiral_restr0.0392420
ELECTRON MICROSCOPYf_plane_restr0.0041694

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