+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-41424 | |||||||||
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Title | Cryo-EM structure of DDB1dB:CRBN:PT-179:SD40, conformation 1 | |||||||||
Map data | main map | |||||||||
Sample |
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Keywords | ubiquitin / CRBN / directed evolution / Zinc finger / IMiD / Molecular glue / TRANSFERASE | |||||||||
Function / homology | Function and homology information negative regulation of large conductance calcium-activated potassium channel activity / 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 / NOTCH3 Intracellular Domain Regulates Transcription / regulation of mitotic cell cycle phase transition ...negative regulation of large conductance calcium-activated potassium channel activity / 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 / NOTCH3 Intracellular Domain Regulates Transcription / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / 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 / viral release from host cell / cullin family protein binding / maltose binding / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / positive regulation of Wnt signaling pathway / ectopic germ cell programmed cell death / negative regulation of protein-containing complex assembly / proteasomal protein catabolic process / positive regulation of viral genome replication / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / pericentric heterochromatin / 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 / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Dual Incision in GG-NER / 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 / site of double-strand break / Neddylation / outer membrane-bounded periplasmic space / protein-macromolecule adaptor activity / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / transmembrane transporter binding / Potential therapeutics for SARS / damaged DNA binding / chromosome, telomeric region / periplasmic space / protein ubiquitination / protein domain specific binding / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA-binding transcription factor activity / DNA repair / negative regulation of DNA-templated transcription / DNA damage response / protein-containing complex binding / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / nucleolus / apoptotic process / perinuclear region of cytoplasm / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.41 Å | |||||||||
Authors | Roy Burman SS / Hunkeler M / Fischer ES | |||||||||
Funding support | United States, 2 items
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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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_41424.map.gz | 63.1 MB | EMDB map data format | |
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Header (meta data) | emd-41424-v30.xml emd-41424.xml | 39.5 KB 39.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_41424_fsc.xml | 10.5 KB | Display | FSC data file |
Images | emd_41424.png | 96.7 KB | ||
Masks | emd_41424_msk_1.map emd_41424_msk_2.map | 125 MB 125 MB | Mask map | |
Filedesc metadata | emd-41424.cif.gz | 9 KB | ||
Others | emd_41424_additional_1.map.gz emd_41424_additional_2.map.gz emd_41424_additional_3.map.gz emd_41424_additional_4.map.gz emd_41424_additional_5.map.gz emd_41424_additional_6.map.gz emd_41424_half_map_1.map.gz emd_41424_half_map_2.map.gz | 3.5 MB 109.4 MB 109.4 MB 115.8 MB 115.8 MB 63.1 MB 115.9 MB 115.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-41424 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-41424 | HTTPS FTP |
-Validation report
Summary document | emd_41424_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_41424_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_41424_validation.xml.gz | 19.3 KB | Display | |
Data in CIF | emd_41424_validation.cif.gz | 24.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-41424 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-41424 | HTTPS FTP |
-Related structure data
Related structure data | 8tnqMC 8tnpC 8tnrC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_41424.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | main map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.8715 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
+Mask #1
+Mask #2
+Additional map: main map, local resolution-filtered
+Additional map: map from CRBN local refinement, post-processed with deepEMhancer
+Additional map: main map, post-processed by deepEMhancer
+Additional map: from local refinement with CRBN mask, half map B
+Additional map: from local refinement with CRBN mask, half map A
+Additional map: from local refinement with CRBN mask
+Half map: half map B
+Half map: half map A
-Sample components
-Entire : Ternary Complex of DDB1dB:CRBN:PT-179 with SD40
Entire | Name: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40 |
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Components |
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-Supramolecule #1: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40
Supramolecule | Name: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Molecular weight | Theoretical: 201 KDa |
-Macromolecule #1: DNA damage-binding protein 1
Macromolecule | Name: DNA damage-binding protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 96.033945 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MGSSHHHHHH SAVDENLYFQ GGGRMSYNYV VTAQKPTAVN GCVTGHFTSA EDLNLLIAKN TRLEIYVVTA EGLRPVKEVG MYGKIAVME LFRPKGESKD LLFILTAKYN ACILEYKQSG ESIDIITRAH GNVQDRIGRP SETGIIGIID PECRMIGLRL Y DGLFKVIP ...String: MGSSHHHHHH SAVDENLYFQ GGGRMSYNYV VTAQKPTAVN GCVTGHFTSA EDLNLLIAKN TRLEIYVVTA EGLRPVKEVG MYGKIAVME LFRPKGESKD LLFILTAKYN ACILEYKQSG ESIDIITRAH GNVQDRIGRP SETGIIGIID PECRMIGLRL Y DGLFKVIP LDRDNKELKA FNIRLEELHV IDVKFLYGCQ APTICFVYQD PQGRHVKTYE VSLREKEFNK GPWKQENVEA EA SMVIAVP EPFGGAIIIG QESITYHNGD KYLAIAPPII KQSTIVCHNR VDPNGSRYLL GDMEGRLFML LLEKEEQMDG TVT LKDLRV ELLGETSIAE CLTYLDNGVV FVGSRLGDSQ LVKLNVDSNE QGSYVVAMET FTNLGPIVDM CVVDLERQGQ GQLV TCSGA FKEGSLRIIR NGIGGNGNSG EIQKLHIRTV PLYESPRKIC YQEVSQCFGV LSSRIEVQDT SGGTTALRPS ASTQA LSSS VSSSKLFSSS TAPHETSFGE EVEVHNLLII DQHTFEVLHA HQFLQNEYAL SLVSCKLGKD PNTYFIVGTA MVYPEE AEP KQGRIVVFQY SDGKLQTVAE KEVKGAVYSM VEFNGKLLAS INSTVRLYEW TTEKELRTEC NHYNNIMALY LKTKGDF IL VGDLMRSVLL LAYKPMEGNF EEIARDFNPN WMSAVEILDD DNFLGAENAF NLFVCQKDSA ATTDEERQHL QEVGLFHL G EFVNVFCHGS LVMQNLGETS TPTQGSVLFG TVNGMIGLVT SLSESWYNLL LDMQNRLNKV IKSVGKIEHS FWRSFHTER KTEPATGFID GDLIESFLDI SRPKMQEVVA NLQYDDGSGM KREATADDLI KVVEELTRIH UniProtKB: DNA damage-binding protein 1, DNA damage-binding protein 1 |
-Macromolecule #2: Protein cereblon
Macromolecule | Name: Protein cereblon / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 55.144594 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MDYKDDDDKS AVDENLYFQG GGRGGSAHIV MVDAYKPTKG GSGMAGEGDQ QDAAHNMGNH LPLLPAESEE EDEMEVEDQD SKEAKKPNI INFDTSLPTS HTYLGADMEE FHGRTLHDDD SCQVIPVLPQ VMMILIPGQT LPLQLFHPQE VSMVRNLIQK D RTFAVLAY ...String: MDYKDDDDKS AVDENLYFQG GGRGGSAHIV MVDAYKPTKG GSGMAGEGDQ QDAAHNMGNH LPLLPAESEE EDEMEVEDQD SKEAKKPNI INFDTSLPTS HTYLGADMEE FHGRTLHDDD SCQVIPVLPQ VMMILIPGQT LPLQLFHPQE VSMVRNLIQK D RTFAVLAY SNVQEREAQF GTTAEIYAYR EEQDFGIEIV KVKAIGRQRF KVLELRTQSD GIQQAKVQIL PECVLPSTMS AV QLESLNK CQIFPSKPVS REDQCSYKWW QKYQKRKFHC ANLTSWPRWL YSLYDAETLM DRIKKQLREW DENLKDDSLP SNP IDFSYR VAACLPIDDV LRIQLLKIGS AIQRLRCELD IMNKCTSLCC KQCQETEITT KNEIFSLSLC GPMAAYVNPH GYVH ETLTV YKACNLNLIG RPSTEHSWFP GYAWTVAQCK ICASHIGWKF TATKKDMSPQ KFWGLTRSAL LPTIPDTEDE ISPDK VILC L UniProtKB: Protein cereblon |
-Macromolecule #3: Maltose/maltodextrin-binding periplasmic protein,SD40
Macromolecule | Name: Maltose/maltodextrin-binding periplasmic protein,SD40 / type: protein_or_peptide / ID: 3 Details: IZKF1/ZFP91 fusion construct that was further engineered to enhance binding of cereblon/DDB1 in the presence of IMiD derivatives Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 50.466012 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGLNDIFEAQ KIEWHEGSSH HHHHHGSSKI EEGKLVIWIN GDKGYNGLAE VGKKFEKDTG IKVTVEHPDK LEEKFPQVAA TGDGPDIIF WAHDRFGGYA QSGLLAEITP DKAFQDKLYP FTWDAVRYNG KLIAYPIAVE ALSLIYNKDL LPNPPKTWEE I PALDKELK ...String: MGLNDIFEAQ KIEWHEGSSH HHHHHGSSKI EEGKLVIWIN GDKGYNGLAE VGKKFEKDTG IKVTVEHPDK LEEKFPQVAA TGDGPDIIF WAHDRFGGYA QSGLLAEITP DKAFQDKLYP FTWDAVRYNG KLIAYPIAVE ALSLIYNKDL LPNPPKTWEE I PALDKELK AKGKSALMFN LQEPYFTWPL IAADGGYAFK YENGKYDIKD VGVDNAGAKA GLTFLVDLIK NKHMNADTDY SI AEAAFNK GETAMTINGP WAWSNIDTSK VNYGVTVLPT FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEA VNKDKP LGAVALKSYE EELAKDPRIA ATMENAQKGE IMPNIPQMSA FWYAVRTAVI NAASGRQTVD EALKDAQTRI TKLE VLFQG PDYKDDDDKS GGGGLLLFCP ICGFTCRQKG NLLRHINLHT GEKLFKYHLY UniProtKB: Maltose/maltodextrin-binding periplasmic protein, DNA-binding protein Ikaros |
-Macromolecule #4: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #5: 2-[(3S)-2,6-dioxopiperidin-3-yl]-5-(morpholin-4-yl)-1H-isoindole-...
Macromolecule | Name: 2-[(3S)-2,6-dioxopiperidin-3-yl]-5-(morpholin-4-yl)-1H-isoindole-1,3(2H)-dione type: ligand / ID: 5 / Number of copies: 1 / Formula: MIQ |
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Molecular weight | Theoretical: 343.334 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.2625 mg/mL | ||||||||||||
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Buffer | pH: 7 Component:
Details: 20 mM HEPES/NaOH pH 7.0, 150 mM NaCl, and 3 mM TCEP. DMSO concentrations were kept below 2% (v/v) | ||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa Details: Grids (Quantifoil UltrAuFoil R 1.2/1.3) were glow discharged in PELCO easiGlow (20 mA, 120s, 39 Pa) | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA EM GP 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.. | ||||||||||||
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. |
-Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 16340 / Average exposure time: 2.497 sec. / Average electron dose: 52.0 e/Å2 Details: Movies (50 frames) collected using beam-shift with 9 holes per stage position (3x3 pattern) |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Details | Phenix real-space refinement without rigid body, with ADP. | ||||||||
Refinement | Space: REAL / Protocol: OTHER / Overall B value: 70 / Target criteria: Cross-correlation coefficient | ||||||||
Output model | PDB-8tnq: |