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- EMDB-41778: Map from local refinement (focused on CRBN) of DDB1dB:CRBN:PT-179... -

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Basic information

Entry
Database: EMDB / ID: EMD-41778
TitleMap from local refinement (focused on CRBN) of DDB1dB:CRBN:PT-179:SD40, conformation 1
Map dataUnsharpened main map
Sample
  • Complex: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: Protein cereblon
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,SD40
Keywordsubiquitin / CRBN / directed evolution / Zinc finger / IMiD / Molecular glue / Ubiquitin ligase / TRANSFERASE
Function / homology
Function and homology information


negative regulation of monoatomic ion transmembrane transport / 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 ...negative regulation of monoatomic ion transmembrane transport / 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 / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / locomotory exploration behavior / negative regulation of reproductive process / negative regulation of developmental process / cullin family protein binding / positive regulation of Wnt signaling pathway / viral release from host cell / ectopic germ cell programmed cell death / positive regulation of viral genome replication / negative regulation of protein-containing complex assembly / positive regulation of gluconeogenesis / proteasomal protein catabolic process / Recognition of DNA damage by PCNA-containing replication complex / nucleotide-excision repair / positive regulation of protein-containing complex assembly / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / regulation of circadian rhythm / 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 / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / Potential therapeutics for SARS / transmembrane transporter binding / damaged DNA binding / chromosome, telomeric region / protein ubiquitination / DNA repair / apoptotic process / DNA damage response / protein-containing complex binding / nucleolus / negative regulation of apoptotic process / perinuclear region of cytoplasm / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / membrane / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Yippee/Mis18/Cereblon / Yippee zinc-binding/DNA-binding /Mis18, centromere assembly / CULT domain / CULT domain profile. / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / Cleavage/polyadenylation specificity factor, A subunit, N-terminal ...Yippee/Mis18/Cereblon / Yippee zinc-binding/DNA-binding /Mis18, centromere assembly / CULT domain / CULT domain profile. / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / PUA-like superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
DNA damage-binding protein 1 / Protein cereblon
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsRoy Burman SS / Hunkeler M / Fischer ES
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA214608 United States
CitationJournal: 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
DepositionAug 29, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41778.png

Downloads & links

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Map

FileDownload / File: emd_41778.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened main map
Voxel sizeX=Y=Z: 0.8715 Å
Density
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.51420677 - 0.8651175
Average (Standard dev.)0.00029444593 (±0.01610714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 278.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41778_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Main map, post-processed with DeepEMhancer

Fileemd_41778_additional_1.map
AnnotationMain map, post-processed with DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Main map, sharpened

Fileemd_41778_additional_2.map
AnnotationMain map, sharpened
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_41778_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_41778_half_map_2.map
Annotationhalf map B
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AxesZYX

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Sample components

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Entire : Ternary Complex of DDB1dB:CRBN:PT-179 with SD40

EntireName: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40
Components
  • Complex: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: Protein cereblon
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,SD40

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Supramolecule #1: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40

SupramoleculeName: Ternary Complex of DDB1dB:CRBN:PT-179 with SD40 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 201 KDa

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Macromolecule #1: DNA damage-binding protein 1

MacromoleculeName: DNA damage-binding protein 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: 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

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Macromolecule #2: Protein cereblon

MacromoleculeName: Protein cereblon / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: 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

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Macromolecule #3: Maltose/maltodextrin-binding periplasmic protein,SD40

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein,SD40 / type: protein_or_peptide / ID: 3
Details: IKZF1/ZFP91 fusion construct that was further engineered to enhance binding of cereblon/DDB1 in the presence of IMiD derivatives
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2625 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
3.0 mMC9H15O6PTCEP

Details: 20 mM HEPES/NaOH pH 7.0, 150 mM NaCl, and 3 mM TCEP. DMSO concentrations were kept below 2% (v/v)
GridModel: 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)
VitrificationCryogen 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..
DetailsDDB1dB_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.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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, two movies per hole) collected using beam-shift with 9 holes per stage position (3x3 pattern)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6798113
Startup modelType of model: OTHER / Details: ab initio in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.0)
Final 3D classificationNumber classes: 10 / Avg.num./class: 169059 / Software - Name: RELION (ver. 4.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.0) / Number images used: 289691
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER

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