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- PDB-9dur: Cryo-EM Structure of CRBN:dHTC1:ENL YEATS -

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Basic information

Entry
Database: PDB / ID: 9dur
TitleCryo-EM Structure of CRBN:dHTC1:ENL YEATS
Components
  • Protein ENL
  • Protein cereblon
KeywordsLIGASE / Mammalian / ENL YEATS / CRBN / dHTC1 / CIP / targeted protein degradation
Function / homology
Function and homology information


negative regulation of monoatomic ion transmembrane transport / Cul4A-RING E3 ubiquitin ligase complex / locomotory exploration behavior / positive regulation of Wnt signaling pathway / negative regulation of protein-containing complex assembly / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / : / RNA Polymerase II Pre-transcription Events / transcription elongation factor complex ...negative regulation of monoatomic ion transmembrane transport / Cul4A-RING E3 ubiquitin ligase complex / locomotory exploration behavior / positive regulation of Wnt signaling pathway / negative regulation of protein-containing complex assembly / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / : / RNA Polymerase II Pre-transcription Events / transcription elongation factor complex / positive regulation of protein-containing complex assembly / fibrillar center / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / transmembrane transporter binding / protein ubiquitination / chromatin binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / nucleoplasm / metal ion binding / nucleus / membrane / cytoplasm / cytosol
Similarity search - Function
AF-9, ANC1 homology domain / : / ANC1 homology domain (AHD) / : / YEATS superfamily / YEATS family / YEATS domain profile. / Yippee/Mis18/Cereblon / Yippee zinc-binding/DNA-binding /Mis18, centromere assembly / CULT domain ...AF-9, ANC1 homology domain / : / ANC1 homology domain (AHD) / : / YEATS superfamily / YEATS family / YEATS domain profile. / Yippee/Mis18/Cereblon / Yippee zinc-binding/DNA-binding /Mis18, centromere assembly / CULT domain / CULT domain profile. / Lon N-terminal domain profile. / Lon protease, N-terminal domain / Lon protease, N-terminal domain superfamily / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily
Similarity search - Domain/homology
: / Protein ENL / Protein cereblon
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsCheong, H. / Hunkeler, M. / Fischer, E.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA214608 United States
CitationJournal: bioRxiv / Year: 2025
Title: High-throughput diversification of protein-ligand surfaces to discover chemical inducers of proximity.
Authors: James B Shaum / Miquel Muñoz I Ordoño / Erica A Steen / Daniela V Wenge / Hakyung Cheong / Moritz Hunkeler / Eric M Bilotta / Zoe Rutter / Paige A Barta / Abby M Thornhill / Natalia ...Authors: James B Shaum / Miquel Muñoz I Ordoño / Erica A Steen / Daniela V Wenge / Hakyung Cheong / Moritz Hunkeler / Eric M Bilotta / Zoe Rutter / Paige A Barta / Abby M Thornhill / Natalia Milosevich / Lauren M Hargis / Jordan Janowski / Timothy R Bishop / Trever R Carter / Bryce da Camara / Matthias Hinterndorfer / Lucas Dada / Wen-Ji He / Fabian Offensperger / Hirotake Furihata / Sydney R Schweber / Charlie Hatton / Yanhe Wen / Benjamin F Cravatt / Keary M Engle / Katherine A Donovan / Bruno Melillo / Seiya Kitamura / Alessio Ciulli / Scott A Armstrong / Eric S Fischer / Georg E Winter / Michael A Erb
Abstract: Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While rational design strategies can expedite the discovery of ...Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4 to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCF , an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.
History
DepositionOct 4, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Protein cereblon
C: Protein ENL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,1124
Polymers73,3702
Non-polymers7422
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein cereblon


Mass: 55144.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N terminal FLAG tagged CRBN / Source: (gene. exp.) Homo sapiens (human) / Gene: CRBN / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96SW2
#2: Protein Protein ENL / YEATS domain-containing protein 1


Mass: 18225.002 Da / Num. of mol.: 1 / Fragment: YEATS domain
Source method: isolated from a genetically manipulated source
Details: C terminal His tag / Source: (gene. exp.) Homo sapiens (human) / Gene: MLLT1, ENL, LTG19, YEATS1 / Production host: Escherichia coli B (bacteria) / References: UniProt: Q03111
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-A1IQT / 2-[3-[2-[4-[[(5~{S})-1,3-bis(oxidanylidene)-2,7-diazaspiro[4.4]nonan-7-yl]sulfonylamino]piperidin-1-yl]ethylcarbamoyl]phenyl]-~{N}-cyclobutyl-imidazo[1,2-a]pyridine-6-carboxamide


Mass: 676.786 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H40N8O6S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Ternary complex of dHTC1 induced DDB1dB/CRBN, ENL YEATS complexCOMPLEXENL YEATS domain bound to full length CRBN bound via degrader dHTC1#1-#20MULTIPLE SOURCES
2ENL YEATS domainCOMPLEX#21RECOMBINANT
3DDB1dB/CRBNCOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.169 MDaNO
220.0182 MDaNO
330.151 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli B (bacteria)37762
43Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.4
Details: 30 mM HEPES/NaOH pH7.4, 150 mM NaCl. DMSO concentrations were kept below 2% (v/v)
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Particles from two grids were merged for this data set. The grids differed in protein concentration. Grid1: DDB1dB/CRBN:dHTC1:ENL YEATS of 1.4, 14, 2.8 uM Grid2: DDB1dB/CRBN:dHTC1:ENL YEATS of 1.6, 16, 3.2 uM
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K
Details: Grids were vitrified using a Leica EM GP plunge freezer operated at 90% humidity and 10 C. Grids were first pre-incubated with 4 uL of 10 uM CRBN-agnostic IKZF1_140-196_Q146A,G151N for 1 ...Details: Grids were vitrified using a Leica EM GP plunge freezer operated at 90% humidity and 10 C. Grids were first pre-incubated with 4 uL of 10 uM CRBN-agnostic IKZF1_140-196_Q146A,G151N for 1 minute and then blotted from behind for 4 s. Immediately, 4 uL of mixturewas applied to the grids before blotting for 4 s and plunging into liquid ethane at -181 C

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.87 sec. / Electron dose: 52 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 22883
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5aparticle selection
2EPU3.7image acquisition
4cryoSPARC4.5.3CTF correction
7ISOLDE1.6.0model fitting
8Coot0.9.8.0model fitting
9UCSF ChimeraX1.6.1model fitting
10Rosetta3.13model fitting
12cryoSPARC4.5.3initial Euler assignment
13cryoSPARC4.5.3final Euler assignment
14cryoSPARC4.5.3classification
15cryoSPARC4.5.33D reconstruction
16PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9955887
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185312 / Symmetry type: POINT
Atomic model buildingB value: 119.8
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
18tnq

8tnq

B8tnqB1
26phy

6phy

A6phyA2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 155.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00324257
ELECTRON MICROSCOPYf_angle_d0.71235775
ELECTRON MICROSCOPYf_chiral_restr0.0463623
ELECTRON MICROSCOPYf_plane_restr0.0054743
ELECTRON MICROSCOPYf_dihedral_angle_d4.9003597

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