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Open data
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Basic information
Entry | Database: PDB / ID: 8tnq | |||||||||
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Title | Cryo-EM structure of DDB1dB:CRBN:PT-179:SD40, conformation 1 | |||||||||
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![]() | TRANSFERASE / ubiquitin / CRBN / directed evolution / Zinc finger / IMiD / Molecular glue | |||||||||
Function / homology | ![]() negative regulation of monoatomic ion transmembrane transport / lymphocyte differentiation / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription ...negative regulation of monoatomic ion transmembrane transport / lymphocyte differentiation / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / detection of maltose stimulus / maltose transport complex / negative regulation of reproductive process / negative regulation of developmental process / locomotory exploration behavior / carbohydrate transport / mesoderm development / cullin family protein binding / carbohydrate transmembrane transporter activity / viral release from host cell / maltose binding / positive regulation of Wnt signaling pathway / maltose transport / maltodextrin transmembrane transport / ectopic germ cell programmed cell death / proteasomal protein catabolic process / positive regulation of viral genome replication / negative regulation of protein-containing complex assembly / pericentric heterochromatin / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / positive regulation of gluconeogenesis / ATP-binding cassette (ABC) transporter complex / erythrocyte differentiation / cell chemotaxis / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / DNA Damage Recognition in GG-NER / positive regulation of protein-containing complex assembly / regulation of circadian rhythm / 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 / chromatin organization / protein-macromolecule adaptor activity / Neddylation / site of double-strand break / outer membrane-bounded periplasmic space / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / transmembrane transporter binding / Potential therapeutics for SARS / chromosome, telomeric region / damaged DNA binding / periplasmic space / protein ubiquitination / cell cycle / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA-binding transcription factor activity / protein domain specific binding / DNA repair / negative regulation of DNA-templated transcription / apoptotic process / DNA damage response / protein-containing complex binding / nucleolus / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / perinuclear region of cytoplasm / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / membrane / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å | |||||||||
![]() | Roy Burman, S.S. / Hunkeler, M. / Fischer, E.S. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Continuous evolution of compact protein degradation tags regulated by selective molecular glues. Authors: Jaron A M Mercer / Stephan J DeCarlo / Shourya S Roy Burman / Vedagopuram Sreekanth / Andrew T Nelson / Moritz Hunkeler / Peter J Chen / Katherine A Donovan / Praveen Kokkonda / Praveen K ...Authors: Jaron A M Mercer / Stephan J DeCarlo / Shourya S Roy Burman / Vedagopuram Sreekanth / Andrew T Nelson / Moritz Hunkeler / Peter J Chen / Katherine A Donovan / Praveen Kokkonda / Praveen K Tiwari / Veronika M Shoba / Arghya Deb / Amit Choudhary / Eric S Fischer / David R Liu / ![]() Abstract: Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of ...Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 592.7 KB | Display | ![]() |
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PDB format | ![]() | 384.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.5 MB | Display | ![]() |
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Full document | ![]() | 1.5 MB | Display | |
Data in XML | ![]() | 48.9 KB | Display | |
Data in CIF | ![]() | 72.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 41424MC ![]() 8tnpC ![]() 8tnrC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 96033.945 Da / Num. of mol.: 1 / Mutation: residues 396-705 deleted Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() | ||||
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#2: Protein | Mass: 55144.594 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() | ||||
#3: Protein | Mass: 50466.012 Da / Num. of mol.: 1 Mutation: engineered to enhance binding of cereblon/DDB1 in the presence of IMiD derivatives Source method: isolated from a genetically manipulated source Details: IZKF1/ZFP91 fusion construct that was further engineered to enhance binding of cereblon/DDB1 in the presence of IMiD derivatives Source: (gene. exp.) ![]() ![]() ![]() Gene: malE, b4034, JW3994, IKZF1, IK1, IKAROS, LYF1, ZNFN1A1 Production host: ![]() ![]() | ||||
#4: Chemical | #5: Chemical | ChemComp-MIQ / | Mass: 343.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H17N3O5 / Feature type: SUBJECT OF INVESTIGATION Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES | ||||||||||||||||||||
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Molecular weight | Value: 0.201 MDa / Experimental value: YES | ||||||||||||||||||||
Buffer solution | pH: 7 Details: 20 mM HEPES/NaOH pH 7.0, 150 mM NaCl, and 3 mM TCEP. DMSO concentrations were kept below 2% (v/v) | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.2625 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: DDB1dB_CRBN, PT-179, and SD40 were mixed and incubated on ice for 1 hour at final concentration of 10.5, 105, and 21 uM, respectively. Then diluted 10-fold before blotting. | ||||||||||||||||||||
Specimen support | Details: Grids (Quantifoil UltrAuFoil R 1.2/1.3) were glow discharged in PELCO easiGlow (20 mA, 120s, 39 Pa) Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K Details: Leica EM-GP plunge freezer with chamber conditions of 10 C and 90% relative humidity. Grids were first pre-incubated with 4 uL of 10 uM CRBN-agnostic IKZF1_140-196_Q146A,G151N for 1 minute ...Details: Leica EM-GP plunge freezer with chamber conditions of 10 C and 90% relative humidity. 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 mixture 1 diluted 10-fold--with the dilution buffer during the 1-minute incubation time--was 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 |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.497 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16340 Details: Movies (50 frames) collected using beam-shift with 9 holes per stage position (3x3 pattern) |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 6798113 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289691 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 70 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient Details: Phenix real-space refinement without rigid body, with ADP. | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 126.02 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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