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- PDB-8uq8: Crystal structure of RNF168 (RING)-UbcH5c fused to H2A-H2B via a ... -

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Basic information

Entry
Database: PDB / ID: 8uq8
TitleCrystal structure of RNF168 (RING)-UbcH5c fused to H2A-H2B via a 2-residue linker
ComponentsE3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
KeywordsTRANSFERASE / RNF168 / UbcH5c / Histone H2A / Histone H2B / Chromatin / Ubiquitin ligase / Ubiquitin-conjugating enzyme / DNA damage response / DNA double-strand break repair / PROTEIN BINDING / PROTEIN BINDING-Transferase 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 / DNA repair-dependent chromatin remodeling / positive regulation of protein targeting to mitochondrion ...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 / DNA repair-dependent chromatin remodeling / positive regulation of protein targeting to mitochondrion / K63-linked polyubiquitin modification-dependent protein binding / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / negative regulation of transcription elongation by RNA polymerase II / protein monoubiquitination / ubiquitin conjugating enzyme activity / protein K63-linked ubiquitination / negative regulation of BMP signaling pathway / protein localization to CENP-A containing chromatin / protein autoubiquitination / protein K48-linked ubiquitination / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / interstrand cross-link repair / SUMOylation of DNA damage response and repair proteins / heterochromatin organization / epigenetic regulation of gene expression / Packaging Of Telomere Ends / ubiquitin ligase complex / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / nucleosome binding / Deposition of new CENPA-containing nucleosomes at the centromere / nucleosomal DNA binding / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / positive regulation of DNA repair / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / TICAM1, RIP1-mediated IKK complex recruitment / DNA methylation / Condensation of Prophase Chromosomes / IKK complex recruitment mediated by RIP1 / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / PRC2 methylates histones and DNA / innate immune response in mucosa / ubiquitin binding / Defective pyroptosis / Negative regulators of DDX58/IFIH1 signaling / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Peroxisomal protein import / Nonhomologous End-Joining (NHEJ) / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Regulation of TNFR1 signaling / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / RING-type E3 ubiquitin transferase / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / protein modification process / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / B-WICH complex positively regulates rRNA expression / G2/M DNA damage checkpoint / Inactivation of CSF3 (G-CSF) signaling / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / Meiotic recombination / RMTs methylate histone arginines / Pre-NOTCH Transcription and Translation / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / double-strand break repair via nonhomologous end joining / Transcriptional regulation of granulopoiesis / protein polyubiquitination / structural constituent of chromatin / antimicrobial humoral immune response mediated by antimicrobial peptide / ubiquitin-protein transferase activity / UCH proteinases / ubiquitin protein ligase activity / nucleosome / double-strand break repair / nucleosome assembly / Antigen processing: Ubiquitination & Proteasome degradation / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Neddylation / site of double-strand break / HATs acetylate histones / Processing of DNA double-strand break ends / histone binding / antibacterial humoral response
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 / Zinc finger RING-type profile. / Zinc finger, RING-type / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Histone H2A type 1-B/E / Ubiquitin-conjugating enzyme E2 D3 / Histone H2B type 2-E / E3 ubiquitin-protein ligase RNF168
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.34 Å
AuthorsHu, Q. / Botuyan, M.V. / 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 23, 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: E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
a: E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,19637
Polymers97,6142
Non-polymers1,58235
Water3,459192
1
A: E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,53116
Polymers48,8071
Non-polymers72415
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
a: E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,66521
Polymers48,8071
Non-polymers85720
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)107.539, 107.539, 113.985
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31

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Components

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Protein , 1 types, 2 molecules Aa

#1: Protein E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E


Mass: 48807.199 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: residues 1-94 of RNF168, followed by residues 2-147 of UbcH5c, followed by residues 33-123 of H2B, followed by residues 12-105 of H2A
Source: (gene. exp.) Homo sapiens (human)
Gene: RNF168, UBE2D3, UBC5C, UBCH5C, H2BC21, H2BFQ, HIST2H2BE, H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE
Production host: Escherichia coli (E. coli)
References: UniProt: Q8IYW5, UniProt: P61077, UniProt: Q16778, UniProt: P04908, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme

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Non-polymers , 5 types, 227 molecules

#2: Chemical...
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: Cl
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Na
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 192 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.89 Å3/Da / Density % sol: 68.41 %
Crystal growTemperature: 288 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Crystals were obtained by mixing 2 microliters of the protein fusion (9.5 mg/mL) in 10 mM HEPES, pH 7.5, 600 mM NaCl, 1 mM TCEP and 2 microliters of the reservoir solution containing 0.1 M ...Details: Crystals were obtained by mixing 2 microliters of the protein fusion (9.5 mg/mL) in 10 mM HEPES, pH 7.5, 600 mM NaCl, 1 mM TCEP and 2 microliters of the reservoir solution containing 0.1 M HEPES, pH 7.5, 2.4 M NaCl

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 25, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.34→50 Å / Num. obs: 61785 / % possible obs: 99.47 % / Redundancy: 7.6 % / Biso Wilson estimate: 34.28 Å2 / CC1/2: 0.998 / CC star: 1 / Rmerge(I) obs: 0.1093 / Rpim(I) all: 0.04232 / Rrim(I) all: 0.1172 / Net I/σ(I): 21.25
Reflection shellResolution: 2.34→2.42 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.8489 / Mean I/σ(I) obs: 4.42 / Num. unique obs: 6078 / CC1/2: 0.895 / CC star: 0.972 / Rpim(I) all: 0.3139 / Rrim(I) all: 0.8489 / % possible all: 98.54

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GB0, 5EGG

4gb0

5egg


Resolution: 2.34→36.06 Å / SU ML: 0.28 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 27.82 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2357 1990 3.23 %
Rwork0.1959 --
obs0.1972 61683 99.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.34→36.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6698 0 60 192 6950
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0046890
X-RAY DIFFRACTIONf_angle_d0.69327
X-RAY DIFFRACTIONf_dihedral_angle_d5.334959
X-RAY DIFFRACTIONf_chiral_restr0.0391040
X-RAY DIFFRACTIONf_plane_restr0.0051193
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.34-2.40.29441290.26114165X-RAY DIFFRACTION98
2.4-2.470.30331440.25134264X-RAY DIFFRACTION100
2.47-2.540.28941460.24524294X-RAY DIFFRACTION99
2.54-2.620.34881480.24324301X-RAY DIFFRACTION100
2.62-2.710.31311330.25354246X-RAY DIFFRACTION100
2.71-2.820.28261520.24254254X-RAY DIFFRACTION100
2.82-2.950.33021400.23124299X-RAY DIFFRACTION100
2.95-3.110.31851520.23814238X-RAY DIFFRACTION100
3.11-3.30.2891400.23194262X-RAY DIFFRACTION100
3.3-3.550.20911420.20064298X-RAY DIFFRACTION100
3.56-3.910.2171410.17424286X-RAY DIFFRACTION100
3.91-4.480.17571480.15334276X-RAY DIFFRACTION100
4.48-5.640.19191390.14984274X-RAY DIFFRACTION100
5.64-36.060.17421360.16364236X-RAY DIFFRACTION99

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