+Open data
-Basic information
Entry | Database: PDB / ID: 8tnp | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of DDB1dB:CRBN:Pomalidomide:SD40 | |||||||||
Components |
| |||||||||
Keywords | TRANSFERASE / ubiquitin / CRBN / directed evolution / Zinc finger / IMiD / Molecular Glue | |||||||||
Function / homology | Function and homology information 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 / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding ...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 / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / NOTCH3 Intracellular Domain Regulates Transcription / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / negative regulation of reproductive process / negative regulation of developmental process / locomotory exploration behavior / cullin family protein binding / mesoderm development / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / positive regulation of Wnt signaling pathway / carbohydrate transport / viral release from host cell / ectopic germ cell programmed cell death / negative regulation of protein-containing complex assembly / positive regulation of viral genome replication / pericentric heterochromatin / positive regulation of gluconeogenesis / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / erythrocyte differentiation / proteasomal protein catabolic process / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / positive regulation of protein-containing complex assembly / DNA Damage Recognition in GG-NER / 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 / protein-macromolecule adaptor activity / site of double-strand break / Neddylation / outer membrane-bounded periplasmic space / chromatin organization / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / Potential therapeutics for SARS / transmembrane transporter binding / 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 / membrane / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Roy Burman, S.S. / Hunkeler, M. / Fischer, E.S. | |||||||||
Funding support | United States, 2items
| |||||||||
Citation | Journal: Science / Year: 2024 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 |
|
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
---|
-Downloads & links
-Download
PDBx/mmCIF format | 8tnp.cif.gz | 486.9 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb8tnp.ent.gz | 324.4 KB | Display | PDB format |
PDBx/mmJSON format | 8tnp.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/tn/8tnp ftp://data.pdbj.org/pub/pdb/validation_reports/tn/8tnp | HTTPS FTP |
---|
-Related structure data
Related structure data | 41423MC 8tnqC 8tnrC C: citing same article (ref.) M: map data used to model this data |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
#1: Protein | Mass: 55144.594 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CRBN / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): HiFive / References: UniProt: Q96SW2 | ||||
---|---|---|---|---|---|
#2: 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.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): HiFive / References: UniProt: Q16531 | ||||
#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.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human) Gene: malE, b4034, JW3994 / Production host: Escherichia coli (E. coli) / Strain (production host): LOBSTR / References: UniProt: P0AEX9, UniProt: Q13422 | ||||
#4: Chemical | #5: Chemical | ChemComp-Y70 / | Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ternary Complex of DDB1dB:CRBN:pomalidomide with SD40 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
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 |
| ||||||||||||||||||||
Specimen | Conc.: 0.2625 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: DDB1dB_CRBN, pomalidomide, 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. |
-Electron microscopy imaging
Microscopy | Model: TFS TALOS |
---|---|
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 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: 4.993 sec. / Electron dose: 53.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2524 Details: Movies (50 frames) collected using beam-shift with 9 holes per stage position (3x3 pattern) |
-Processing
EM software |
| ||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1254659 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55205 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 102 / Protocol: OTHER / Space: REAL / Target criteria: CC / Details: Phenix real-space refinement without rigid body | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
| ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 129.03 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
|