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Yorodumi- PDB-8u13: Cryo-EM structure of the human nucleosome core particle ubiquityl... -
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-Basic information
Entry | Database: PDB / ID: 8u13 | |||||||||
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Title | Cryo-EM structure of the human nucleosome core particle ubiquitylated at histone H2A lysine 15 in complex with RNF168-UbcH5c (class 1) | |||||||||
Components |
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Keywords | STRUCTURAL PROTEIN/DNA/TRANSFERASE / Nucleosome core particle / chromatin / RNF168 / UbcH5c / DNA repair / DNA double-strand break / Homologous recombination / BRCA1-BARD1 / 53BP1 / ubiquitin / STRUCTURAL PROTEIN-DNA-TRANSFERASE complex | |||||||||
Function / homology | Function and homology information histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / isotype switching / double-strand break repair via classical nonhomologous end joining / DNA repair-dependent chromatin remodeling / K63-linked polyubiquitin modification-dependent protein binding / response to ionizing radiation / negative regulation of transcription elongation by RNA polymerase II / protein K63-linked ubiquitination / nucleosome binding ...histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / isotype switching / double-strand break repair via classical nonhomologous end joining / DNA repair-dependent chromatin remodeling / K63-linked polyubiquitin modification-dependent protein binding / response to ionizing radiation / negative regulation of transcription elongation by RNA polymerase II / protein K63-linked ubiquitination / nucleosome binding / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / interstrand cross-link repair / CENP-A containing nucleosome / ubiquitin ligase complex / epigenetic regulation of gene expression / SUMOylation of DNA damage response and repair proteins / 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 / positive regulation of DNA repair / Inhibition of DNA recombination at telomere / Meiotic synapsis / telomere organization / RNA Polymerase I Promoter Opening / Interleukin-7 signaling / SUMOylation of chromatin organization proteins / Assembly of the ORC complex at the origin of replication / DNA methylation / Condensation of Prophase Chromosomes / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / innate immune response in mucosa / PRC2 methylates histones and DNA / ubiquitin binding / Defective pyroptosis / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RING-type E3 ubiquitin transferase / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / G2/M DNA damage checkpoint / B-WICH complex positively regulates rRNA expression / HDMs demethylate histones / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / double-strand break repair via nonhomologous end joining / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / ubiquitin-protein transferase activity / UCH proteinases / double-strand break repair / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / site of double-strand break / gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / Factors involved in megakaryocyte development and platelet production / Processing of DNA double-strand break ends / HATs acetylate histones / antibacterial humoral response / histone binding / ubiquitin-dependent protein catabolic process / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / chromosome, telomeric region / protein ubiquitination / Ub-specific processing proteases / defense response to Gram-positive bacterium / cadherin binding / Amyloid fiber formation / protein heterodimerization activity / negative regulation of cell population proliferation / DNA damage response / chromatin binding / protein-containing complex / DNA binding / extracellular space Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Hu, Q. / Botuyan, M.V. / Zhao, D. / Cui, G. / Mer, G. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8u13.cif.gz | 351.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8u13.ent.gz | 263.1 KB | Display | PDB format |
PDBx/mmJSON format | 8u13.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u1/8u13 ftp://data.pdbj.org/pub/pdb/validation_reports/u1/8u13 | HTTPS FTP |
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-Related structure data
Related structure data | 41800MC 8smwC 8smxC 8smyC 8smzC 8sn0C 8sn1C 8sn2C 8sn3C 8sn4C 8sn5C 8sn6C 8sn7C 8sn8C 8sn9C 8snaC 8txvC 8txwC 8txxC 8u14C 8upfC 8uq8C 8uq9C 8uqaC 8uqbC 8uqcC 8uqdC 8uqeC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 5 types, 9 molecules AEBFDHKCG
#1: Protein | 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 | 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 | Mass: 14084.348 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) Gene: H2BC4, H2BFL, HIST1H2BC, H2BC6, H2BFH, HIST1H2BE, H2BC7, H2BFG, HIST1H2BF, H2BC8, H2BFA, HIST1H2BG, H2BC10, H2BFK, HIST1H2BI Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P62807 #6: Protein | | Mass: 11175.100 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RNF168 / Plasmid: pHISPP / Production host: Escherichia coli (E. coli) References: UniProt: Q8IYW5, RING-type E3 ubiquitin transferase #7: Protein | Mass: 12992.091 Da / Num. of mol.: 2 / Mutation: K13S Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE / Plasmid: pHISPP / Production host: Escherichia coli (E. coli) / References: UniProt: P04908 |
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-DNA chain , 2 types, 2 molecules IJ
#4: DNA chain | Mass: 45138.770 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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#5: DNA chain | 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) |
-Non-polymers , 1 types, 2 molecules
#8: Chemical |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Human nucleosome core particle ubiquitylated at histone H2A lysine 15 in complex with RNF168-UbcH5c (class 1) Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.255 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 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 support | Grid material: COPPER / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5697 Details: 5697 images were recorded in movie mode. 5503 were retained for particle picking. |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 9989935 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84664 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Type: experimental model
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Refine LS restraints |
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