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- PDB-6xmx: Cryo-EM structure of BCL6 bound to BI-3802 -

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Basic information

Entry
Database: PDB / ID: 6xmx
TitleCryo-EM structure of BCL6 bound to BI-3802
ComponentsB-cell lymphoma 6 protein
KeywordsTRANSCRIPTION / transcription factor / degrader
Function / homology
Function and homology information


regulation of memory T cell differentiation / negative regulation of mitotic cell cycle DNA replication / intronic transcription regulatory region sequence-specific DNA binding / negative regulation of isotype switching to IgE isotypes / negative regulation of plasma cell differentiation / negative regulation of T-helper 2 cell differentiation / isotype switching to IgE isotypes / negative regulation of mast cell cytokine production / regulation of germinal center formation / plasma cell differentiation ...regulation of memory T cell differentiation / negative regulation of mitotic cell cycle DNA replication / intronic transcription regulatory region sequence-specific DNA binding / negative regulation of isotype switching to IgE isotypes / negative regulation of plasma cell differentiation / negative regulation of T-helper 2 cell differentiation / isotype switching to IgE isotypes / negative regulation of mast cell cytokine production / regulation of germinal center formation / plasma cell differentiation / paraspeckles / germinal center formation / negative regulation of leukocyte proliferation / pyramidal neuron differentiation / regulation of immune system process / type 2 immune response / positive regulation of regulatory T cell differentiation / T-helper 2 cell differentiation / negative regulation of B cell apoptotic process / positive regulation of cell motility / negative regulation of Rho protein signal transduction / erythrocyte development / FOXO-mediated transcription of cell death genes / negative regulation of cell-matrix adhesion / regulation of T cell proliferation / negative regulation of Notch signaling pathway / TP53 regulates transcription of several additional cell death genes whose specific roles in p53-dependent apoptosis remain uncertain / B cell proliferation / regulation of cell differentiation / negative regulation of cellular senescence / Rho protein signal transduction / regulation of immune response / heterochromatin formation / positive regulation of B cell proliferation / regulation of cytokine production / positive regulation of neuron differentiation / cell-matrix adhesion / transcription corepressor binding / cell motility / cell morphogenesis / protein localization / negative regulation of cell growth / chromatin DNA binding / DNA-binding transcription repressor activity, RNA polymerase II-specific / sequence-specific double-stranded DNA binding / regulation of cell population proliferation / regulation of inflammatory response / actin cytoskeleton organization / spermatogenesis / Interleukin-4 and Interleukin-13 signaling / DNA-binding transcription factor binding / sequence-specific DNA binding / transcription by RNA polymerase II / inflammatory response / positive regulation of apoptotic process / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA damage response / chromatin binding / nucleolus / Golgi apparatus / negative regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding / metal ion binding / nucleus
Similarity search - Function
BTB/POZ domain / BTB domain profile. / Zinc finger, C2H2 type / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / zinc finger / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 superfamily / SKP1/BTB/POZ domain superfamily / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type
Similarity search - Domain/homology
Chem-U52 / B-cell lymphoma 6 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsYoon, H. / Burman, S.S.R. / Hunkeler, M. / Nowak, R.P. / Fischer, E.S.
Funding support United States, Switzerland, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA218278 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA218278 United States
Swiss National Science Foundation174331 Switzerland
The Mark Foundation United States
CitationJournal: Nature / Year: 2020
Title: Small-molecule-induced polymerization triggers degradation of BCL6.
Authors: Mikołaj Słabicki / Hojong Yoon / Jonas Koeppel / Lena Nitsch / Shourya S Roy Burman / Cristina Di Genua / Katherine A Donovan / Adam S Sperling / Moritz Hunkeler / Jonathan M Tsai / Rohan ...Authors: Mikołaj Słabicki / Hojong Yoon / Jonas Koeppel / Lena Nitsch / Shourya S Roy Burman / Cristina Di Genua / Katherine A Donovan / Adam S Sperling / Moritz Hunkeler / Jonathan M Tsai / Rohan Sharma / Andrew Guirguis / Charles Zou / Priya Chudasama / Jessica A Gasser / Peter G Miller / Claudia Scholl / Stefan Fröhling / Radosław P Nowak / Eric S Fischer / Benjamin L Ebert /
Abstract: Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy ...Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets, but a substantial subset of proteins are resistant to targeted degradation using existing approaches. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.
History
DepositionJul 1, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 25, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: 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 1.2Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: B-cell lymphoma 6 protein
B: B-cell lymphoma 6 protein
C: B-cell lymphoma 6 protein
D: B-cell lymphoma 6 protein
E: B-cell lymphoma 6 protein
F: B-cell lymphoma 6 protein
G: B-cell lymphoma 6 protein
H: B-cell lymphoma 6 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)359,92216
Polymers356,0428
Non-polymers3,8808
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, significant shift in Mw upon addition of compound, microscopy, negative stain EM clearly reveals polymerized species, microscopy, cryo-EM was used to solve the structure of the assembly
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
B-cell lymphoma 6 protein / / BCL-6 / B-cell lymphoma 5 protein / BCL-5 / Protein LAZ-3 / Zinc finger and BTB domain-containing ...BCL-6 / B-cell lymphoma 5 protein / BCL-5 / Protein LAZ-3 / Zinc finger and BTB domain-containing protein 27 / Zinc finger protein 51


Mass: 44505.305 Da / Num. of mol.: 8 / Mutation: R160C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BCL6, BCL5, LAZ3, ZBTB27, ZNF51 / Plasmid: pAC-derived / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P41182
#2: Chemical
ChemComp-U52 / 2-[6-[[5-chloranyl-2-[(3~{S},5~{R})-3,5-dimethylpiperidin-1-yl]pyrimidin-4-yl]amino]-1-methyl-2-oxidanylidene-quinolin-3-yl]oxy-~{N}-methyl-ethanamide


Mass: 484.978 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C24H29ClN6O3 / 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: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: B-cell lymphoma 6 protein (BCL6) filament / Type: ORGANELLE OR CELLULAR COMPONENT
Details: BCL6 polymerized upon addition of small molecule BI-3802. 4 dimers enclosed in map.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.352 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHepesC8H18N2O4S1
2200 mMSodium chlorideNaClSodium chloride1
31 mMTCEPC9H15O6P1
SpecimenConc.: 0.48 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Strep II-Avi BCL6 (aa5-360) polymerized by addition of 1.5 molar excess BI-3802. CHAPSO (0.8 mM final) added for grid preparation.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K / Details: 4 ul sample applied twice, blotted 1.3s each time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Data collection in counting mode, using multi shot scheme (4 holes per stage position, 2 shots per hole)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1000 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: 3 sec. / Electron dose: 63.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7553
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
5RELIONCTF correction
8Coot0.89model fitting
10PHENIX1.17model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
Image processingDetails: The selected movies were corrected for beam-induced motion.
CTF correctionDetails: Standard CTF correction in relion, based on parameters estimated by CTFFIND4
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1610413 / Details: particles picked with crYOLO
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112048 / Details: as implemented in Relion / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Real_space_refine: global minimization, rigid body and adp refinement with target restraints.
Atomic model buildingPDB-ID: 5MW2

5mw2


Pdb chain-ID: A / Accession code: 5MW2 / Pdb chain residue range: 7-128 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 23.38 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01238208
ELECTRON MICROSCOPYf_angle_d1.096111096
ELECTRON MICROSCOPYf_chiral_restr0.0571296
ELECTRON MICROSCOPYf_plane_restr0.00561368
ELECTRON MICROSCOPYf_dihedral_angle_d20.52293088

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