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- PDB-8tnp: Cryo-EM structure of DDB1dB:CRBN:Pomalidomide:SD40 -

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Basic information

Entry
Database: PDB / ID: 8tnp
TitleCryo-EM structure of DDB1dB:CRBN:Pomalidomide:SD40
Components
  • DNA damage-binding protein 1
  • Maltose/maltodextrin-binding periplasmic protein,SD40
  • Protein cereblon
KeywordsTRANSFERASE / ubiquitin / CRBN / directed evolution / Zinc finger / IMiD / Molecular Glue
Function / homology
Function and homology information


lymphocyte differentiation / negative regulation of monoatomic ion transmembrane transport / positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / NOTCH3 Intracellular Domain Regulates Transcription / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding ...lymphocyte differentiation / negative regulation of monoatomic ion transmembrane transport / positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / NOTCH3 Intracellular Domain Regulates Transcription / 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 / detection of maltose stimulus / maltose transport complex / negative regulation of reproductive process / mesoderm development / negative regulation of developmental process / carbohydrate transport / locomotory exploration behavior / cullin family protein binding / viral release from host cell / carbohydrate transmembrane transporter activity / maltose binding / positive regulation of Wnt signaling pathway / maltose transport / maltodextrin transmembrane transport / ectopic germ cell programmed cell death / negative regulation of protein-containing complex assembly / positive regulation of viral genome replication / pericentric heterochromatin / proteasomal protein catabolic process / 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 / 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 / Formation of Incision Complex in GG-NER / Wnt signaling pathway / 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 / outer membrane-bounded periplasmic space / chromatin organization / 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 / periplasmic space / chromosome, telomeric region / protein ubiquitination / 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 / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / protein-containing complex binding / nucleolus / perinuclear region of cytoplasm / protein-containing complex / extracellular space / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / membrane / cytoplasm / cytosol
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 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / zinc finger / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / 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
S-Pomalidomide / Maltose/maltodextrin-binding periplasmic protein / DNA-binding protein Ikaros / DNA damage-binding protein 1 / Protein cereblon
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRoy Burman, S.S. / Hunkeler, M. / Fischer, E.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA214608 United States
Cancer Research Institute 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 2, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation_author
Revision 1.2Apr 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Protein cereblon
A: DNA damage-binding protein 1
C: Maltose/maltodextrin-binding periplasmic protein,SD40
hetero molecules


Theoretical massNumber of molelcules
Total (without water)202,0496
Polymers201,6453
Non-polymers4043
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein cereblon


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 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 / 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 Maltose/maltodextrin-binding periplasmic protein,SD40


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 ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-Y70 / S-Pomalidomide


Mass: 273.244 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H11N3O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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:pomalidomide with SD40 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.201 MDa / Experimental value: YES
Buffer solutionpH: 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
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
33 mMTCEPC9H15O6P1
SpecimenConc.: 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 supportDetails: 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
VitrificationInstrument: 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

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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)

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2SerialEM4.1bimage acquisition
4cryoSPARC4.2.0CTF correction
7UCSF ChimeraX1.5model fitting
9cryoSPARC4.2.0initial Euler assignment
10cryoSPARC4.2.0final Euler assignment
11cryoSPARC4.2.0classification
12cryoSPARC4.2.03D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1254659
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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 buildingB 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

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
15FQD

5fqd

B5FQDB1
28G46

8g46

A8G46A2
36H0F

6h0f

C6H0FC3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 129.03 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00459419
ELECTRON MICROSCOPYf_angle_d0.547912742
ELECTRON MICROSCOPYf_chiral_restr0.0451441
ELECTRON MICROSCOPYf_plane_restr0.00381629
ELECTRON MICROSCOPYf_dihedral_angle_d4.88781249

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