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- PDB-8ov6: Ternary structure of intramolecular bivalent glue degrader IBG1 b... -

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Basic information

Entry
Database: PDB / ID: 8ov6
TitleTernary structure of intramolecular bivalent glue degrader IBG1 bound to BRD4 and DCAF16:DDB1deltaBPB
Components
  • Bromodomain-containing protein 4
  • DDB1- and CUL4-associated factor 16
  • DDB1deltaBPB
KeywordsTRANSCRIPTION / Bromodomain / BET protein / DCAF16 / Bivalent glue / Targeted protein degradation / TPD
Function / homology
Function and homology information


Cul4-RING E3 ubiquitin ligase complex / RNA polymerase II C-terminal domain binding / negative regulation of DNA damage checkpoint / P-TEFb complex binding / negative regulation by host of viral transcription / positive regulation of T-helper 17 cell lineage commitment / positive regulation of G2/M transition of mitotic cell cycle / histone reader activity / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome ...Cul4-RING E3 ubiquitin ligase complex / RNA polymerase II C-terminal domain binding / negative regulation of DNA damage checkpoint / P-TEFb complex binding / negative regulation by host of viral transcription / positive regulation of T-helper 17 cell lineage commitment / positive regulation of G2/M transition of mitotic cell cycle / histone reader activity / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome / positive regulation of transcription elongation by RNA polymerase II / transcription coregulator activity / lysine-acetylated histone binding / p53 binding / Neddylation / chromosome / regulation of inflammatory response / positive regulation of canonical NF-kappaB signal transduction / Potential therapeutics for SARS / transcription coactivator activity / transcription cis-regulatory region binding / protein ubiquitination / chromatin remodeling / DNA damage response / chromatin binding / chromatin / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / enzyme binding / positive regulation of transcription by RNA polymerase II / nucleoplasm / nucleus
Similarity search - Function
DDB1- and CUL4-associated factor 16 / DDB1- and CUL4-associated factor 16 / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / NET domain superfamily / NET domain profile. / Brdt, bromodomain, repeat II / Brdt, bromodomain, repeat I / NET domain / Bromodomain extra-terminal - transcription regulation ...DDB1- and CUL4-associated factor 16 / DDB1- and CUL4-associated factor 16 / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / NET domain superfamily / NET domain profile. / Brdt, bromodomain, repeat II / Brdt, bromodomain, repeat I / NET domain / Bromodomain extra-terminal - transcription regulation / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / Bromodomain profile. / bromo domain / Bromodomain / Bromodomain-like superfamily
Similarity search - Domain/homology
Chem-U79 / Bromodomain-containing protein 4 / DDB1- and CUL4-associated factor 16
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsCowan, A.D. / Sundaramoorthy, R. / Nakasone, M.A. / Ciulli, A.
Funding supportEuropean Union, Switzerland, United Kingdom, Austria, 9items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission101024945European Union
European Research Council (ERC)ERC-2012-StG-311460European Union
Innovative Medicines Initiative875510 Switzerland
Wellcome Trust223816/Z/21/Z United Kingdom
Medical Research Council (MRC, United Kingdom)4050845509 United Kingdom
European Research Council (ERC)851478European Union
Austrian Science FundP32125 Austria
Austrian Science FundP31690 Austria
Austrian Science FundP7909 Austria
Citation
Journal: Nature / Year: 2024
Title: Targeted protein degradation via intramolecular bivalent glues.
Authors: Oliver Hsia / Matthias Hinterndorfer / Angus D Cowan / Kentaro Iso / Tasuku Ishida / Ramasubramanian Sundaramoorthy / Mark A Nakasone / Hana Imrichova / Caroline Schätz / Andrea Rukavina / ...Authors: Oliver Hsia / Matthias Hinterndorfer / Angus D Cowan / Kentaro Iso / Tasuku Ishida / Ramasubramanian Sundaramoorthy / Mark A Nakasone / Hana Imrichova / Caroline Schätz / Andrea Rukavina / Koraljka Husnjak / Martin Wegner / Alejandro Correa-Sáez / Conner Craigon / Ryan Casement / Chiara Maniaci / Andrea Testa / Manuel Kaulich / Ivan Dikic / Georg E Winter / Alessio Ciulli /
Abstract: Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal ...Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
#1: Journal: Biorxiv / Year: 2023
Title: Targeted protein degradation via intramolecular bivalent glues
Authors: Hsia, O. / Hinterndorfer, M. / Cowan, A.D. / Iso, K. / Ishida, I. / Sundaramoorthy, R. / Nakasone, M.A. / Imrichova, H. / Schatz, C. / Rukvina, A. / Husnjak, K. / Wegner, M. / Correa-Saez, A. ...Authors: Hsia, O. / Hinterndorfer, M. / Cowan, A.D. / Iso, K. / Ishida, I. / Sundaramoorthy, R. / Nakasone, M.A. / Imrichova, H. / Schatz, C. / Rukvina, A. / Husnjak, K. / Wegner, M. / Correa-Saez, A. / Craigon, C. / Casement, R. / Maniaci, C. / Testa, A. / Kaulich, M. / Dikic, I. / Winter, G.E. / Ciulli, A.
History
DepositionApr 25, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Jan 10, 2024Group: Database references / Refinement description / Structure summary
Category: citation / citation_author ...citation / citation_author / em_3d_fitting_list / struct
Item: _citation.title / _em_3d_fitting_list.initial_refinement_model_id / _struct.title
Revision 1.3Feb 21, 2024Group: Database references / Category: citation / citation_author
Revision 1.4Mar 6, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.5Mar 13, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DDB1deltaBPB
B: DDB1- and CUL4-associated factor 16
C: Bromodomain-containing protein 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,7725
Polymers164,8843
Non-polymers8882
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, DCAF16:DDB1deltaBPB were co-expressed in insect cells, BRD4 was expressed in E. coli. The complex with compound 1 was assembled in vitro and purified by gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7060 Å2
ΔGint-30 kcal/mol
Surface area53280 Å2

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Components

#1: Protein DDB1deltaBPB


Mass: 93347.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1 / Production host: Trichoplusia ni (cabbage looper)
#2: Protein DDB1- and CUL4-associated factor 16


Mass: 24333.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DCAF16, C4orf30 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NXF7
#3: Protein Bromodomain-containing protein 4 / Protein HUNK1


Mass: 47203.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRD4, HUNK1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O60885
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-U79 / methyl 2-[(9~{S})-7-[4-[4-[[4-[(3-cyano-4-methyl-1~{H}-indol-7-yl)sulfamoyl]phenyl]methylcarbamoyl]phenyl]phenyl]-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^{2,6}]trideca-2(6),4,7,10,12-pentaen-9-yl]ethanoate


Mass: 822.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C44H38N8O5S2

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Ternary complex of bivalent glue degrader compound 1 bound to BRD4 and DCAF16:DDB1deltaBPBCOMPLEXDCAF16:DDB1deltaBPB were co-expressed in insect cells, BRD4 was expressed in E. coli. The complex with compound 1 was assembled in vitro and purified by gel filtration#1-#30RECOMBINANT
2DCAF16:DDB1deltaBPB E3 ligase substrate receptor:apator complexCOMPLEX#1-#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.165 MDaNO
210.118 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
30.5 mMTCEPC9H15O6P1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The components were co-incubated then loaded onto a 10/300 Superdex 200 gl column and the fraction corresponding to the complex was collected and concentrated to 0.8 mg/mL
Specimen supportDetails: Current 35 mA / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3.5 uL complex was dispensed onto the grid, allowed to disperse for 10 s, blotted for 3.5 s using blot force 3, then plunged into liquid ethane

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Calibrated magnification: 190000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 12.7 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4x)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC4.1.2particle selection
2EPU3image acquisition
4cryoSPARC4.1.2CTF correction
7PHENIX1.20.1-4487model fitting
9cryoSPARC4.1.2initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
12cryoSPARC3D reconstruction
13PHENIX1.20.1-4487model refinement
14UCSF ChimeraX1.6.dev202302040126model refinement
15ISOLDE1.6.dev1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192014 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeDetails
15FQD

5fqd

5FQD1PDBexperimental model
23MXF

3mxf

3MXF2PDBexperimental model
36DUV

6duv

6DUV3PDBexperimental model
4OtherotherDCAF16 model was derived from both colabfold and model-angelo
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 100.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00259485
ELECTRON MICROSCOPYf_angle_d0.504712848
ELECTRON MICROSCOPYf_chiral_restr0.04181441
ELECTRON MICROSCOPYf_plane_restr0.00421662
ELECTRON MICROSCOPYf_dihedral_angle_d4.93861299

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