EMDB-62509: cryo-EM structure of RNF20/RNF40-RAD6A-Ub in complex with H2BS112...

Basic information

Entry

Database: EMDB / ID: EMD-62509

• Title: cryo-EM structure of RNF20/RNF40-RAD6A-Ub in complex with H2BS112GlcNAc nucleosome

Sample

• Complex: RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome
  • Protein or peptide: x 8 types
  • DNA: x 2 types
• Ligand: x 2 types

• Keywords: RNF20 / RNF40 / H2BS112GlcNAc / O-GlcNAcylation / ubiquitination / H2BK120Ub / NUCLEAR PROTEIN

Function / homology

Function:

histone H2B C-terminal K residue ubiquitin ligase activity / HULC complex / polytene chromosome / positive regulation of mitophagy / protein K11-linked ubiquitination / DNA damage tolerance / RHOBTB1 GTPase cycle / E2 ubiquitin-conjugating enzyme / ubiquitin conjugating enzyme activity / ubiquitin ligase complex / response to UV / protein K48-linked ubiquitination / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / 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 / telomere organization / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / RNA Polymerase I Promoter Opening / negative regulation of cell migration / Inhibition of DNA recombination at telomere / Assembly of the ORC complex at the origin of replication / Meiotic synapsis / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / ubiquitin binding / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / innate immune response in mucosa / Defective pyroptosis / mRNA 3'-UTR binding / Negative Regulation of CDH1 Gene Transcription / HDACs deacetylate histones / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / 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 / HDMs demethylate histones / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / G2/M DNA damage checkpoint / NoRC negatively regulates rRNA expression / RING-type E3 ubiquitin transferase / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / DNA Damage/Telomere Stress Induced Senescence / Pre-NOTCH Transcription and Translation / Meiotic recombination / G2/M transition of mitotic cell cycle / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Metalloprotease DUBs / Transcriptional regulation of granulopoiesis / RMTs methylate histone arginines / protein polyubiquitination / HCMV Early Events / p53 binding / ubiquitin-protein transferase activity / structural constituent of chromatin / ubiquitin protein ligase activity / UCH proteinases / late endosome / heterochromatin formation / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / Antigen processing: Ubiquitination & Proteasome degradation / nucleosome assembly / antibacterial humoral response / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / chromatin organization / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / histone binding / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / killing of cells of another organism / ubiquitin-dependent protein catabolic process / defense response to Gram-negative bacterium / proteasome-mediated ubiquitin-dependent protein catabolic process / transcription coactivator activity / mitochondrial outer membrane / chromosome, telomeric region / lysosome / Ub-specific processing proteases

Domain/homology:

E3 ubiquitin ligase Bre1 / : / 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 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 / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily

Component:

Histone H3 / Ubiquitin B / Histone H2B type 1-K / E3 ubiquitin-protein ligase BRE1B / Histone H2A type 1-B/E / Ubiquitin-conjugating enzyme E2 A / Histone H4 / E3 ubiquitin-protein ligase BRE1A

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.48 Å
• Authors: Deng ZH / Ai HS / Liu L

Funding support

China, 1 items

Organization	Grant number	Country
National Natural Science Foundation of China (NSFC)	22137005, 92253302, 22227810 and T2488301, to L.L.; 32501108, to H.A.; 22207065, to Y.L.; 22277073, to M.P	China

• Citation: Journal: Nat Chem Biol / Year: 2026; Title: Allosteric activation of RNF20/RNF40-RAD6A-mediated H2BK120 monoubiquitylation by H2BS112 GlcNAcylation.; Authors: Zhiheng Deng / Shixian Tao / Yunxiang Du / Yulei Li / Liying Zhang / Qiang Shi / Xiaoru Du / Maoshen Sun / Zebin Tong / Man Pan / Lei Liu / Huasong Ai / ; Abstract: The activation of H2B K120 monoubiquitylation (H2BK120ub) by H2B S112 GlcNAcylation (H2BS112GlcNAc) has an important role in regulating transcriptional activation, yet its mechanism remains unclear. Here we chemically synthesized H2BS112GlcNAc-modified nucleosomes and quantitatively evaluated how H2BS112GlcNAc stimulates ubiquitylation by RNF20/RNF40-RAD6A E3-E2 enzymes. Cryo-electron microscopy determination of a chemically trapped RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex revealed that the H2BS112GlcNAc moiety interacts with the E2 enzyme RAD6A but not the E3 ligase RNF20/RNF40. Mutagenesis and kinetics analyses demonstrated that H2BS112GlcNAc allosterically stimulates ubiquitin transfer from the RAD6A~Ub thioester to H2B K120 by enhancing the nucleophilicity of H2B K120. Structure‒activity relationship analysis further identified the essential roles of the C2 N-acetyl group and the β-configuration of C1 on the H2BS112GlcNAc moiety. These findings provide the structural evidence of histone posttranslational modification crosstalk involving O-GlcNAcylation and reveal how O-GlcNAcylation can allosterically stimulate enzyme activity through substrate modification.

History

Deposition	Nov 26, 2024	-
Header (metadata) release	Dec 31, 2025	-
Map release	Dec 31, 2025	-
Update	Feb 4, 2026	-
Current status	Feb 4, 2026	Processing site: PDBj / Status: Released

Map

• File: Download / File: emd_62509.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.074 Å

Density

Contour Level	By AUTHOR: 0.0045
Minimum - Maximum	-0.07828837 - 0.13465728
Average (Standard dev.)	0.000028244322 (±0.0037853764)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 274.944 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_62509_msk_1.map

Additional map: Conformation 3 of the dynamic analysis

• File: emd_62509_additional_1.map
• Annotation: Conformation 3 of the dynamic analysis

Additional map: Conformation 2 of the dynamic analysis

• File: emd_62509_additional_2.map
• Annotation: Conformation 2 of the dynamic analysis

Additional map: Conformation 1 of the dynamic analysis

• File: emd_62509_additional_3.map
• Annotation: Conformation 1 of the dynamic analysis

Half map: #1

• File: emd_62509_half_map_1.map

Half map: #2

• File: emd_62509_half_map_2.map

Sample components

Entire : RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome

• Entire: Name: RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome

Components

• Complex: RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome
  • Protein or peptide: E3 ubiquitin-protein ligase BRE1B
  • Protein or peptide: Polyubiquitin-B
  • Protein or peptide: Histone H2A type 1-B/E
  • Protein or peptide: Histone H2B type 1-K
  • Protein or peptide: Histone H3
  • Protein or peptide: Histone H4
  • DNA: DNA (147-MER)
  • DNA: DNA (147-MER)
  • Protein or peptide: E3 ubiquitin-protein ligase BRE1A
  • Protein or peptide: Ubiquitin-conjugating enzyme E2 A
• Ligand: ZINC ION
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

Supramolecule #1: RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome

• Supramolecule: Name: RNF20/RNF40-RAD6A-Ub bound to H2BS112GlcNAc nucleosome; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: E3 ubiquitin-protein ligase BRE1B

• Macromolecule: Name: E3 ubiquitin-protein ligase BRE1B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 7.120409 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

EYKARLTCPC CNTRKKDAVL TKCFHVFCFE CVRGRYEARQ RKCPKCNAAF GAHDFHRIYI

UniProtKB: E3 ubiquitin-protein ligase BRE1B

Macromolecule #2: Polyubiquitin-B

• Macromolecule: Name: Polyubiquitin-B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 8.519778 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG

UniProtKB: Ubiquitin B

Macromolecule #3: Histone H2A type 1-B/E

• Macromolecule: Name: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 14.034355 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

SGRGKQGGKA RAKAKTRSSR AGLQFPVGRV HRLLRKGNYS ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRN DEELNKLLGR VTIAQGGVLP NIQAVLLPKK TESHHKAKGK

UniProtKB: Histone H2A type 1-B/E

Macromolecule #4: Histone H2B type 1-K

• Macromolecule: Name: Histone H2B type 1-K / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 13.76398 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHP DTGISSKAMG IMNSFVNDIF ERIAGEASRL AHYNKRSTI TSREIQTAVR LLLPGELAKH AVSEGTKAVT CYTSAK

UniProtKB: Histone H2B type 1-K

Macromolecule #5: Histone H3

• Macromolecule: Name: Histone H3 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 15.273838 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAV MALQEASEAY LVGLFEDTNL SAIHAKRVTI MPKDIQLARR IRGERA

UniProtKB: Histone H3

Macromolecule #6: Histone H4

• Macromolecule: Name: Histone H4 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 11.263231 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYA LKRQGRTLYG FGG

UniProtKB: Histone H4

Macromolecule #9: E3 ubiquitin-protein ligase BRE1A

• Macromolecule: Name: E3 ubiquitin-protein ligase BRE1A / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 7.14447 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

DYKARLTCPC CNMRKKDAVL TKCFHVFCFE CVKTRYDTRQ RKCPKCNAAF GANDFHRIYI

UniProtKB: E3 ubiquitin-protein ligase BRE1A

Macromolecule #10: Ubiquitin-conjugating enzyme E2 A

• Macromolecule: Name: Ubiquitin-conjugating enzyme E2 A / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 17.114129 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MSTPARRRLM RDFKRLQEDP PAGVSGAPSE NNIMVWNAVI FGPEGTPFED GTFKLTIEFT EEYPNKPPTV RFVSKMFHPN VYADGSICL DILQNRWSPT YDVSSILTSI QSLLDEPNPN SPANSQAAQL YQENKREYEK RVSAIVEQSW R

UniProtKB: Ubiquitin-conjugating enzyme E2 A

Macromolecule #7: DNA (147-MER)

• Macromolecule: Name: DNA (147-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 45.604047 KDa

Sequence

String:

(DA)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC) (DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG) (DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG) (DA) (DG)(DT)(DA)(DA)(DT)(DC)(DC)(DC) (DC)(DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT) (DA)(DA) (DA)(DA)(DC)(DG)(DC)(DG)(DG) (DG)(DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DC) (DG)(DT)(DA) (DC)(DG)(DT)(DG)(DC)(DG) (DT)(DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT) (DG)(DC)(DT)(DA) (DG)(DA)(DG)(DC)(DT) (DG)(DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC) (DA)(DA)(DT)(DT)(DG) (DA)(DG)(DC)(DG) (DG)(DC)(DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC) (DC)(DG)(DG)(DG)(DA)(DT) (DT)(DC)(DT) (DC)(DC)(DA)(DG)

Macromolecule #8: DNA (147-MER)

• Macromolecule: Name: DNA (147-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 45.145754 KDa

Sequence

String:

(DC)(DT)(DG)(DG)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA) (DA)(DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA) (DG) (DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT) (DA)(DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT) (DA)(DA) (DA)(DC)(DG)(DC)(DA)(DC)(DG) (DT)(DA)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT) (DC)(DC)(DC) (DC)(DC)(DG)(DC)(DG)(DT) (DT)(DT)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DC) (DA)(DA)(DG)(DG) (DG)(DG)(DA)(DT)(DT) (DA)(DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG)(DT) (DC)(DT)(DC)(DC)(DA) (DG)(DG)(DC)(DA) (DC)(DG)(DT)(DG)(DT)(DC)(DA)(DG)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DC) (DA)(DT)(DC) (DC)(DT)(DG)(DT)

Macromolecule #11: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 11 / Number of copies: 4 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Macromolecule #12: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 12 / Number of copies: 1 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5
• Vitrification: Cryogen name: ETHANE

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 18.0 µm / Nominal defocus min: 1.0 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing #1

• Image processing ID: 1
• CTF correction: Type: NONE
• Startup model: Type of model: INSILICO MODEL
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 119602
• Initial angle assignment: Type: RANDOM ASSIGNMENT
• Final angle assignment: Type: ANGULAR RECONSTITUTION

Image processing #2

• Image processing ID: 2
• CTF correction: Type: NONE
• Startup model: Type of model: OTHER
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 119602
• Initial angle assignment: Type: RANDOM ASSIGNMENT
• Final angle assignment: Type: ANGULAR RECONSTITUTION