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- EMDB-60259: conformation 3 cryo-EM map of N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-60259
Titleconformation 3 cryo-EM map of N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2 at a resolution of 8.33 angstrom
Map data
Sample
  • Complex: N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2
KeywordsCullin1 / CDC34 / ubiquitin / Skp1 / Skp2 / Cks1 / LIGASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.33 Å
AuthorsAi HS / Liang LJ / Li CT / Zhao FY / Zhang LY / Li JH
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Am Chem Soc / Year: 2025
Title: Design and Semisynthesis of Ubiquitin Extension Probes for Structural Analysis of Cullin1-Mediated Substrate Polyubiquitination.
Authors: Chuntong Li / Fangyu Zhao / Chong Zuo / Liying Zhang / Yangwode Jing / Xu Li / Guo-Chao Chu / Luyu Shi / Yingyue Zhang / Han Wang / Shuzhe Sun / Maoshen Sun / Huasong Ai / Lu-Jun Liang / Jinghong Li /
Abstract: Chemical trapping strategies have recently emerged as powerful approaches for investigating the structural dynamics of E3 ligase-catalyzed substrate ubiquitination. However, current ubiquitination- ...Chemical trapping strategies have recently emerged as powerful approaches for investigating the structural dynamics of E3 ligase-catalyzed substrate ubiquitination. However, current ubiquitination-derived probes are limited to studying substrate mono- or diubiquitination events. Probes capable of investigating how E3 ligases accommodate E2-Ub conjugates and ubiquitinated substrates to generate longer ubiquitin chains remain unexplored. In this work, we report the development of two Cullin1 E3 ligase (CRL1)-dependent probes, Extension Probe and Extension Probe, which mimic transient intermediates formed during CRL1-catalyzed K48-linked diubiquitin and tetraubiquitin chain formation on substrate p27. Notably, a chemoenzymatic semisynthetic strategy was devised to generate Extension Probe, involving the enzymatic conjugation of a preformed K48-linked diubiquitin to a synthetic Ub-p27-degron construct using the E2 conjugating enzyme UBE2K. Both Extension Probe and Extension Probe formed stable complexes with N8-CRL1 (comprising neddylated Cullin1-Rbx1 and the substrate receptor complex Skp1-Skp2-Cks1), facilitating structural analysis by chemical cross-linking mass spectrometry (CX-MS) and cryo-electron microscopy (cryo-EM). Our results indicate the presence of multiple distinct conformations of the catalytic module (comprising the RING domain of Rbx1, CDC34-Ub, and the acceptor ubiquitin) within the di- and tetraubiquitination complexes, while the conformation of the Cullin1-Skp1-Skp2-Cks1 subunit remains unchanged. In conclusion, this work expands the toolkit available for chemical trapping strategies and provides advanced insights into CRL-catalyzed substrate polyubiquitination.
History
DepositionMay 23, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_60259.png

Downloads & links

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Map

FileDownload / File: emd_60259.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.074 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.0031140891 - 0.02635531
Average (Standard dev.)0.0001808009 (±0.0012481675)
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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Half map: #1

Fileemd_60259_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Half map: #2

Fileemd_60259_half_map_2.map
Projections & Slices
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Sample components

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Entire : N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2

EntireName: N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2
Components
  • Complex: N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2

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Supramolecule #1: N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2

SupramoleculeName: N8_Cullin1/Rbx1/Skp1/Skp2/Cks2/p27 in complex with Extension Probe-Ub2
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

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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: FLOOD BEAM / 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

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 64010
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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