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- EMDB-47729: Cryo-EM structure of CSN-N8CUL1 complex with CSN5i-3 -

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Basic information

Entry
Database: EMDB / ID: EMD-47729
TitleCryo-EM structure of CSN-N8CUL1 complex with CSN5i-3
Map data
Sample
  • Complex: The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3
KeywordsCOP9 / COP9 signalosome / signalosome / deneddylation / CSN5i-3 / orthosteric molecule glue / inhibitor / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsShi H / Zheng N
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2026
Title: CSN5i-3 is an orthosteric molecular glue inhibitor of COP9 signalosome.
Authors: Huigang Shi / Xiaorong Wang / Clinton Yu / Haibin Mao / Fenglong Jiao / Merav Braitbard / Ben Shor / Zhongsheng Zhang / Thomas R Hinds / Shiyun Cao / Erkang Fan / Dina Schneidman-Duhovny / ...Authors: Huigang Shi / Xiaorong Wang / Clinton Yu / Haibin Mao / Fenglong Jiao / Merav Braitbard / Ben Shor / Zhongsheng Zhang / Thomas R Hinds / Shiyun Cao / Erkang Fan / Dina Schneidman-Duhovny / Lan Huang / Ning Zheng /
Abstract: Orthosteric inhibitors block enzyme active sites and prevent substrates from binding. Enhancing their specificity through substrate dependence seems inherently unlikely, as their mechanism hinges on ...Orthosteric inhibitors block enzyme active sites and prevent substrates from binding. Enhancing their specificity through substrate dependence seems inherently unlikely, as their mechanism hinges on direct competition rather than selective recognition. Here we show that a molecular glue mechanism unexpectedly imparts substrate-dependent potency to CSN5i-3, an orthosteric inhibitor of the COP9 signalosome (CSN). We first confirm that CSN5i-3 inhibits CSN, which catalyses NEDD8 (N8) deconjugation from the cullin-RING ubiquitin ligases, by occupying the active site of its catalytic subunit, CSN5, and directly competing with the iso-peptide bond substrate. Notably, the orthosteric inhibitor binds free CSN with only micromolar affinity, yet achieves nanomolar potency in blocking its deneddylase activity. Cryogenic electron microscopy structures of the enzyme-substrate-inhibitor complex reveal that active site-engaged CSN5i-3 occludes the substrate iso-peptide linkage while simultaneously extending an N8-binding exosite of CSN5, acting as a molecular glue to cement the N8-CSN5 interaction. The cooperativity of this trimolecular CSN5i-3-N8-CSN5 assembly, in turn, sequesters CSN5i-3 at its binding site, conferring high potency to the orthosteric inhibitor despite its low affinity for the free enzyme. Together, our findings highlight the modest affinity requirements of molecule glues for individual target proteins and establish orthosteric molecular glue inhibitors as a new class of substrate-dependent enzyme antagonists.
History
DepositionNov 6, 2024-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateFeb 25, 2026-
Current statusFeb 25, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47729.png

Downloads & links

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Map

FileDownload / File: emd_47729.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 330.8 Å		0.83 Å/pix.
x 400 pix.
= 330.8 Å		0.83 Å/pix.
x 400 pix.
= 330.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.8924658 - 1.4911146
Average (Standard dev.)0.00048965606 (±0.030024605)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 330.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47729_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_47729_half_map_1.map
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Half map: #2

Fileemd_47729_half_map_2.map
Projections & Slices
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Sample components

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Entire : The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3

EntireName: The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3
Components
  • Complex: The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3

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Supramolecule #1: The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3

SupramoleculeName: The COP9 signalosome in complex with neddylated cullin-1 and CSN5i-3
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
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 174577
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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