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- EMDB-35379: Bre1(FL)-Rad6-nucleosome complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35379
TitleBre1(FL)-Rad6-nucleosome complex
Map dataE3-E2-NCP
Sample
  • Complex: Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome
KeywordsNucleosome / Bre1 / Rad6 / NUCLEAR PROTEIN
Biological speciesSaccharomyces (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsAi H / Deng Z / Pan M / Liu L
Funding support China, 1 items
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, 2023-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35379.png

Downloads & links

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Map

FileDownload / File: emd_35379.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE3-E2-NCP
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.074 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.009915374 - 0.03798305
Average (Standard dev.)0.00024305595 (±0.0018368157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.944 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: E3-NCP

Fileemd_35379_additional_1.map
AnnotationE3-NCP
Projections & Slices
AxesZYX

Projections

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Density Histograms

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Half map: #1

Fileemd_35379_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35379_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome

EntireName: Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome
Components
  • Complex: Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome

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Supramolecule #1: Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome

SupramoleculeName: Complex of full-length Bre1 and Rad6-Ub bound to the nucleosome
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces (fungus)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 17255
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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