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- PDB-9nwt: Cryo-EM structure of DDB1dB:CRBN:mezigdomide:SALL4(392-449;G416A) -

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Basic information

Entry
Database: PDB / ID: 9nwt
TitleCryo-EM structure of DDB1dB:CRBN:mezigdomide:SALL4(392-449;G416A)
Components
  • DNA damage-binding protein 1
  • Protein cereblon
  • Sal-like protein 4
KeywordsLIGASE / DDB1 / cereblon / mezigdomide / SALL4
Function / homology
Function and homology information


POU5F1 (OCT4), SOX2, NANOG activate genes related to proliferation / Transcriptional regulation of pluripotent stem cells / negative regulation of monoatomic ion transmembrane transport / positive regulation by virus of viral protein levels in host cell / embryonic limb morphogenesis / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / ventricular septum development / inner cell mass cell proliferation ...POU5F1 (OCT4), SOX2, NANOG activate genes related to proliferation / Transcriptional regulation of pluripotent stem cells / negative regulation of monoatomic ion transmembrane transport / positive regulation by virus of viral protein levels in host cell / embryonic limb morphogenesis / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / ventricular septum development / inner cell mass cell proliferation / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of reproductive process / negative regulation of developmental process / locomotory exploration behavior / viral release from host cell / cullin family protein binding / somatic stem cell population maintenance / positive regulation of Wnt signaling pathway / ectopic germ cell programmed cell death / positive regulation of viral genome replication / negative regulation of protein-containing complex assembly / heterochromatin / proteasomal protein catabolic process / positive regulation of gluconeogenesis / Regulation of PTEN gene transcription / nucleotide-excision repair / neural tube closure / positive regulation of protein-containing complex assembly / Recognition of DNA damage by PCNA-containing replication complex / regulation of circadian rhythm / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Wnt signaling pathway / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / site of double-strand break / Neddylation / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / transmembrane transporter binding / DNA-binding transcription factor activity, RNA polymerase II-specific / chromosome, telomeric region / protein ubiquitination / DNA repair / intracellular membrane-bounded organelle / apoptotic process / DNA damage response / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / protein-containing complex binding / nucleolus / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / protein-containing complex / extracellular space / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / metal ion binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
: / 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) ...: / 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) / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / PUA-like superfamily / Zinc finger, C2H2 type / zinc finger / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 superfamily / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Mezigdomide / DNA damage-binding protein 1 / Protein cereblon / Sal-like protein 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsPark, J. / Hunkeler, M. / Roy Burman, S.S. / Fischer, E.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA214608 United States
CitationJournal: Mol Cell / Year: 2025
Title: Expanding the druggable zinc-finger proteome defines properties of drug-induced degradation.
Authors: Mikołaj Słabicki / Jiho Park / Radosław P Nowak / Shourya S Roy Burman / Jesse Pellman / Charles Zou / Hlib Razumkov / Jeannie Carreiro / Simran Rastogi / Anna Goldstein / Marek M Nagiec ...Authors: Mikołaj Słabicki / Jiho Park / Radosław P Nowak / Shourya S Roy Burman / Jesse Pellman / Charles Zou / Hlib Razumkov / Jeannie Carreiro / Simran Rastogi / Anna Goldstein / Marek M Nagiec / Katherine A Donovan / Jianwei Che / Moritz Hunkeler / Qixiang Geng / Chi-Lin Hsu / Megha Lakshminarayan / Chelsea Shu / Rebecca L Zon / Zuzanna Kozicka / Paul M C Park / Jonathan M Tsai / Hojong Yoon / Lyn H Jones / Adam S Sperling / Nathanael S Gray / Eric S Fischer / Benjamin L Ebert /
Abstract: Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4 to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has ...Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4 to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design.
History
DepositionMar 24, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Sal-like protein 4
A: DNA damage-binding protein 1
B: Protein cereblon
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,3856
Polymers162,6863
Non-polymers6983
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Sal-like protein 4 / Zinc finger protein 797 / Zinc finger protein SALL4


Mass: 11507.800 Da / Num. of mol.: 1 / Mutation: G416A
Source method: isolated from a genetically manipulated source
Details: StrepII-Avi-TEV-SALL4(392-449;G416A) / Source: (gene. exp.) Homo sapiens (human) / Gene: SALL4, ZNF797 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJQ4
#2: Protein DNA damage-binding protein 1 / DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / ...DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / HBV X-associated protein 1 / XAP-1 / UV-damaged DNA-binding factor / UV-damaged DNA-binding protein 1 / UV-DDB 1 / XPE-binding factor / XPE-BF / Xeroderma pigmentosum group E-complementing protein / XPCe


Mass: 96033.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396- ...Details: His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16531
#3: Protein Protein cereblon


Mass: 55144.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: FLAG-Spy-TEV-CRBN(1-442) / Source: (gene. exp.) Homo sapiens (human) / Gene: CRBN / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96SW2
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-QFC / Mezigdomide


Mass: 567.610 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C32H30FN5O4 / 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

ComponentName: Ternary complex of DDB1dB:CRBN:mezigdomide:SALL4(392-449;G416A)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: High Five
Buffer solutionpH: 7.4 / Details: 50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 3 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES/NaOH pH 7.4C8H18N2O4S/NaOH1
2150 mMNaClNaCl1
33 mMTCEPC9H15O6P1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 10 uM His6-DDB1dB:FLAG-Spy-CRBN, 100 uM mezigdomide, and 20 uM StrepII-Avi-SALL4(392-449;G416A) were mixed in dilution buffer (50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 3 mM TCEP) and incubated ...Details: 10 uM His6-DDB1dB:FLAG-Spy-CRBN, 100 uM mezigdomide, and 20 uM StrepII-Avi-SALL4(392-449;G416A) were mixed in dilution buffer (50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 3 mM TCEP) and incubated for 1 hr. Additional dilution buffer was added to the mixture to achieve final concentrations of 1.6 uM DDB1dB:CRBN, 16.0 uM mezigdomide, and 3.2 uM SALL4(392-449;G416A), and 4 uL of the diluted mixture were applied to the grid.
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: UltrAuFoil R 0.6/1 300 mesh grids were glow-discharged for 2 min at 20 mA and 39 Pa, pre-incubated with 4 uL of 10 uM FLAG-IKZF1(140-196;Q146A/G151N) for 1 min, and blotted from behind for 4 ...Details: UltrAuFoil R 0.6/1 300 mesh grids were glow-discharged for 2 min at 20 mA and 39 Pa, pre-incubated with 4 uL of 10 uM FLAG-IKZF1(140-196;Q146A/G151N) for 1 min, and blotted from behind for 4 s. 4 uL of the sample was then applied to the grid. Grids were vitrified using an EM GP plunge freezer operated at 90% humidity and 10 C with 0 s pre-blot, 4 s blot, and 0 s post-blot.

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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: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.8 sec. / Electron dose: 55.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12502
Details: One movie was recorded per hole with 25 holes per stage position
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2SerialEM4.1bimage acquisition
4cryoSPARC4.2.0CTF correction
7UCSF ChimeraX1.6.1model fitting
9cryoSPARC4.2.0initial Euler assignment
10cryoSPARC4.2.0final Euler assignment
11cryoSPARC4.2.0classification
12cryoSPARC4.2.03D reconstruction
13PHENIX1.21-5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9479675
Details: Particles were picked using Topaz (v0.2.5) trained on templates from on-the-fly 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 479953 / Algorithm: FOURIER SPACE
Details: Particles were re-extracted at 0.83 A/pixel, and subsequently processed by reference-based motion correction and global CTF refinement (per-group beam tilt and trefoil). Homogeneous ...Details: Particles were re-extracted at 0.83 A/pixel, and subsequently processed by reference-based motion correction and global CTF refinement (per-group beam tilt and trefoil). Homogeneous refinement of these processed particles followed by local refinement with a mask encompassing the full particle yielded a final reconstruction at 2.7 A. A local refinement using the CRBN:drug:ZF-focused mask further improved drug and ZF density.
Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: Sharpened and unsharpened maps processed by cryoSPARC, in addition to maps post-processed with DeepEMhancer (v0.16), were used for model building. A previously-built model of DDB1dB:CRBN: ...Details: Sharpened and unsharpened maps processed by cryoSPARC, in addition to maps post-processed with DeepEMhancer (v0.16), were used for model building. A previously-built model of DDB1dB:CRBN:mezigdomide:SALL4(392-449) was fit into the density as individual chains using ChimeraX (v1.6.1). Using the same ligand restraint file described above, the model was relaxed into the density using Rosetta (v3.13) followed by manual adjustment in Coot. The model was prepared for refinement using phenix.ready_set (v1.21-5207) and refined using phenix.real_space_refine (v1.21-5207).
Atomic model building

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

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

8tnq

A8TNQA1
28TNQ

8tnq

B8TNQB1
38U17

8u17

C8U17C2
48D7Z

8d7z

D8D7ZD3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 131.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00189852
ELECTRON MICROSCOPYf_angle_d0.418813335
ELECTRON MICROSCOPYf_chiral_restr0.04361502
ELECTRON MICROSCOPYf_plane_restr0.00691718
ELECTRON MICROSCOPYf_dihedral_angle_d8.42363692

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