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- PDB-8iej: RNF20-RNF40/hRad6A-Ub/nucleosome complex -

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Entry
Database: PDB / ID: 8iej
TitleRNF20-RNF40/hRad6A-Ub/nucleosome complex
Components
  • (DNA (147-MER)) x 2
  • (E3 ubiquitin-protein ligase ...) x 2
  • (Histone H2B type 1- ...) x 2
  • Histone H2A type 1-B/E
  • Histone H3.1
  • Histone H4
  • Ubiquitin-conjugating enzyme E2 A
KeywordsNUCLEAR PROTEIN / RNF20 / RFN40 / RAD6 / nucleosome / H2BK120Ub
Function / homology
Function and homology information


histone H2B C-terminal K residue ubiquitin ligase activity / HULC complex / positive regulation of mitophagy / protein K11-linked ubiquitination / postreplication repair / RHOBTB1 GTPase cycle / E2 ubiquitin-conjugating enzyme / ubiquitin conjugating enzyme activity / ubiquitin ligase complex / negative regulation of megakaryocyte differentiation ...histone H2B C-terminal K residue ubiquitin ligase activity / HULC complex / positive regulation of mitophagy / protein K11-linked ubiquitination / postreplication repair / RHOBTB1 GTPase cycle / E2 ubiquitin-conjugating enzyme / ubiquitin conjugating enzyme activity / ubiquitin ligase complex / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / protein K48-linked ubiquitination / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / response to UV / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Inhibition of DNA recombination at telomere / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / 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 / DNA methylation / Condensation of Prophase Chromosomes / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / negative regulation of cell migration / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / epigenetic regulation of gene expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / ubiquitin binding / chloroplast / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / mRNA 3'-UTR binding / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / 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 / RING-type E3 ubiquitin transferase / DNA Damage/Telomere Stress Induced Senescence / 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 / HCMV Early Events / protein polyubiquitination / ubiquitin-protein transferase activity / G2/M transition of mitotic cell cycle / structural constituent of chromatin / p53 binding / UCH proteinases / ubiquitin protein ligase activity / nucleosome / late endosome / Antigen processing: Ubiquitination & Proteasome degradation / heterochromatin formation / E3 ubiquitin ligases ubiquitinate target proteins / nucleosome assembly / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / ubiquitin-dependent protein catabolic process / small ribosomal subunit rRNA binding / Oxidative Stress Induced Senescence / gene expression / Estrogen-dependent gene expression / histone binding / proteasome-mediated ubiquitin-dependent protein catabolic process / transcription coactivator activity / chromosome, telomeric region / lysosome / neuron projection / Ub-specific processing proteases
Similarity search - Function
E3 ubiquitin ligase Bre1 / : / Histone, subunit A / Histone, subunit A / 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 / Ubiquitin-conjugating enzyme ...E3 ubiquitin ligase Bre1 / : / Histone, subunit A / Histone, subunit A / 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 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Histone H2A conserved site / Histone H2A signature. / 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. / Ribosomal protein S6, plastid/chloroplast / Zinc finger, RING-type / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Translation elongation factor EF1B/ribosomal protein S6 / Histone-fold / Zinc finger, RING/FYVE/PHD-type / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Small ribosomal subunit protein bS6c alpha / E3 ubiquitin-protein ligase BRE1B / Histone H2A type 1-B/E / Ubiquitin-conjugating enzyme E2 A / Histone H4 / Histone H3.1 / E3 ubiquitin-protein ligase BRE1A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsAi, H. / Deng, Z. / Sun, M. / Du, Y. / Pan, M. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)22137005, 92253302, 22227810, 22277073, 92253302 China
CitationJournal: Mol Cell / Year: 2023
Title: Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.
Authors: Zhiheng Deng / Huasong Ai / Maoshen Sun / Zebin Tong / Yunxiang Du / Qian Qu / Liying Zhang / Ziyu Xu / Shixian Tao / Qiang Shi / Jia-Bin Li / Man Pan / Lei Liu /
Abstract: Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme ...Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.
History
DepositionFeb 15, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Histone H2A type 1-B/E
D: Histone H2B type 1-K
E: Histone H3.1
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-K
I: DNA (147-MER)
J: DNA (147-MER)
K: Histone H3.1
L: Histone H4
A: E3 ubiquitin-protein ligase BRE1B
M: E3 ubiquitin-protein ligase BRE1A
R: Ubiquitin-conjugating enzyme E2 A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,56717
Polymers208,30513
Non-polymers2624
Water00
1


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

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Components

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Protein , 4 types, 7 molecules CGEKFLR

#1: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 11936.948 Da / Num. of mol.: 2
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
#3: 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: 11562.577 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
Production host: Escherichia coli (E. coli) / References: UniProt: P68431
#4: Protein Histone H4


Mass: 9123.692 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, H4C16, H4-16, HIST4H4
Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#10: Protein Ubiquitin-conjugating enzyme E2 A / E2 ubiquitin-conjugating enzyme A / RAD6 homolog A / HR6A / hHR6A / Ubiquitin carrier protein A / ...E2 ubiquitin-conjugating enzyme A / RAD6 homolog A / HR6A / hHR6A / Ubiquitin carrier protein A / Ubiquitin-protein ligase A


Mass: 17114.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2A, RAD6A / Production host: Escherichia coli (E. coli)
References: UniProt: P49459, E2 ubiquitin-conjugating enzyme

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Histone H2B type 1- ... , 2 types, 2 molecules DH

#2: Protein Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 10451.957 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC12, H2BFT, HIRIP1, HIST1H2BK / Production host: Escherichia coli (E. coli) / References: UniProt: O60814
#5: Protein Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 10477.994 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC12, H2BFT, HIRIP1, HIST1H2BK / Production host: Escherichia coli (E. coli) / References: UniProt: O60814

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DNA chain , 2 types, 2 molecules IJ

#6: DNA chain DNA (147-MER)


Mass: 45604.047 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)
#7: DNA chain DNA (147-MER)


Mass: 45145.754 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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E3 ubiquitin-protein ligase ... , 2 types, 2 molecules AM

#8: Protein E3 ubiquitin-protein ligase BRE1B / BRE1-B / 95 kDa retinoblastoma-associated protein / RBP95 / RING finger protein 40 / RING-type E3 ...BRE1-B / 95 kDa retinoblastoma-associated protein / RBP95 / RING finger protein 40 / RING-type E3 ubiquitin transferase BRE1B


Mass: 7120.409 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF40, BRE1B, KIAA0661
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
References: UniProt: O75150, RING-type E3 ubiquitin transferase
#9: Protein E3 ubiquitin-protein ligase BRE1A / BRE1-A / hBRE1 / RING finger protein 20 / RING-type E3 ubiquitin transferase BRE1A


Mass: 7144.470 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF20, BRE1A
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
References: UniProt: Q5VTR2, RING-type E3 ubiquitin transferase

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Non-polymers , 1 types, 4 molecules

#11: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / 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: RNF20-RNF40/hRAD6A-Ub in complex with nucleosome / Type: COMPLEX / Entity ID: #1-#10 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 239429 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515138
ELECTRON MICROSCOPYf_angle_d0.61221703
ELECTRON MICROSCOPYf_dihedral_angle_d30.1114109
ELECTRON MICROSCOPYf_chiral_restr0.0392438
ELECTRON MICROSCOPYf_plane_restr0.0051745

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