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Entry
Database: PDB / ID: 8sn2
TitleCryo-EM structure of the human nucleosome core particle in complex with RNF168 and UbcH5c (UbcH5c chemically conjugated to histone H2A)
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 / 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 / protein K6-linked ubiquitination / Signaling by BMP / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / E2 ubiquitin-conjugating enzyme ...histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / (E3-independent) E2 ubiquitin-conjugating enzyme / protein K6-linked ubiquitination / Signaling by BMP / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / E2 ubiquitin-conjugating enzyme / K63-linked polyubiquitin modification-dependent protein binding / response to ionizing radiation / DNA repair-dependent chromatin remodeling / ubiquitin conjugating enzyme activity / negative regulation of transcription elongation by RNA polymerase II / negative regulation of BMP signaling pathway / protein K63-linked ubiquitination / negative regulation of tumor necrosis factor-mediated signaling pathway / protein monoubiquitination / ubiquitin ligase complex / negative regulation of megakaryocyte differentiation / interstrand cross-link repair / protein localization to CENP-A containing chromatin / protein K48-linked ubiquitination / SUMOylation of DNA damage response and repair proteins / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / nucleosome binding / 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 / 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 / Inhibition of DNA recombination at telomere / telomere organization / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / positive regulation of DNA repair / Assembly of the ORC complex at the origin of replication / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / SUMOylation of chromatin organization proteins / 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 / IKK complex recruitment mediated by RIP1 / HCMV Late Events / innate immune response in mucosa / ubiquitin binding / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PINK1-PRKN Mediated Mitophagy / PRC2 methylates histones and DNA / epigenetic regulation of gene expression / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Negative regulators of DDX58/IFIH1 signaling / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Peroxisomal protein import / lipopolysaccharide binding / Regulation of TNFR1 signaling / Transcriptional regulation by small RNAs / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / NoRC negatively regulates rRNA expression / protein modification process / Inactivation of CSF3 (G-CSF) signaling / RING-type E3 ubiquitin transferase / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / PKMTs methylate histone lysines / Meiotic recombination / double-strand break repair via nonhomologous end joining / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / heterochromatin formation / nucleosome assembly / protein polyubiquitination / ubiquitin-protein transferase activity / antimicrobial humoral immune response mediated by antimicrobial peptide / structural constituent of chromatin / UCH proteinases / ubiquitin protein ligase activity / antibacterial humoral response
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 H2A / Histone 2A / 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 / Histone H3 signature 1. / Zinc finger RING-type profile. / Histone H3 signature 2. / Zinc finger, RING-type / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / 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: 3.6 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
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
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,09114
Polymers228,96012
Non-polymers1312
Water00
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 , 6 types, 10 molecules AEBFCGDHKL

#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 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 / Plasmid: pHISPP / Production host: Escherichia coli (E. coli)
References: UniProt: P61077, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme

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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: 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: 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: C-flat-1.2/1.3
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: 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: 11501
Details: 11501 images were recorded in movie-mode of which 11333 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
13cryoSPARC4.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 12545249
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22664 / 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

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