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- EMDB-22265: Cryo-EM structure of BCL6 bound to BI-3802 -

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Basic information

Entry
Database: EMDB / ID: EMD-22265
TitleCryo-EM structure of BCL6 bound to BI-3802
Map dataMain map
Sample
  • Organelle or cellular component: B-cell lymphoma 6 protein (BCL6) filament
    • Protein or peptide: B-cell lymphoma 6 protein
  • Ligand: 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
Keywordstranscription factor / degrader / TRANSCRIPTION
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
B-cell lymphoma 6 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsYoon H / Burman SSR
Funding support United States, Switzerland, 4 items
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, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xmx
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22265.png

Downloads & links

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Map

FileDownload / File: emd_22265.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.04927374 - 0.085074484
Average (Standard dev.)0.00016575813 (±0.002001609)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8250.8250.825
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z211.200211.200211.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0490.0850.000

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Supplemental data

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Mask #1

Fileemd_22265_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half map (1) from refinement

Fileemd_22265_half_map_1.map
Annotationunfiltered half map (1) from refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half map (2) from refinement

Fileemd_22265_half_map_2.map
Annotationunfiltered half map (2) from refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : B-cell lymphoma 6 protein (BCL6) filament

EntireName: B-cell lymphoma 6 protein (BCL6) filament
Components
  • Organelle or cellular component: B-cell lymphoma 6 protein (BCL6) filament
    • Protein or peptide: B-cell lymphoma 6 protein
  • Ligand: 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

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Supramolecule #1: B-cell lymphoma 6 protein (BCL6) filament

SupramoleculeName: B-cell lymphoma 6 protein (BCL6) filament / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: BCL6 polymerized upon addition of small molecule BI-3802. 4 dimers enclosed in map.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 352 KDa

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Macromolecule #1: B-cell lymphoma 6 protein

MacromoleculeName: B-cell lymphoma 6 protein / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.505305 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDWSHPQFEK SAVGLNDIFE AQKIEWHEGG GGSGENLYFQ GGGRADSCIQ FTRHASDVLL NLNRLRSRDI LTDVVIVVSR EQFRAHKTV LMACSGLFYS IFTDQLKCNL SVINLDPEIN PEGFCILLDF MYTSRLNLRE GNIMAVMATA MYLQMEHVVD T CRKFIKAS ...String:
MDWSHPQFEK SAVGLNDIFE AQKIEWHEGG GGSGENLYFQ GGGRADSCIQ FTRHASDVLL NLNRLRSRDI LTDVVIVVSR EQFRAHKTV LMACSGLFYS IFTDQLKCNL SVINLDPEIN PEGFCILLDF MYTSRLNLRE GNIMAVMATA MYLQMEHVVD T CRKFIKAS EAEMVSAIKP PREEFLNSRM LMPQDIMAYR GCEVVENNLP LRSAPGCESR AFAPSLYSGL STPPASYSMY SH LPVSSLL FSDEEFRDVR MPVANPFPKE RALPCDSARP VPGEYSRPTL EVSPNVCHSN IYSPKETIPE EARSDMHYSV AEG LKPAAP SARNAPYFPC DKASKEEERP SSEDEIALHF EPPNAPLNRK GLVSPQSPQK SDCQPNSPTE SCSSKNACIL QASG

UniProtKB: B-cell lymphoma 6 protein

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Macromolecule #2: 2-[6-[[5-chloranyl-2-[(3~{S},5~{R})-3,5-dimethylpiperidin-1-yl]py...

MacromoleculeName: 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
type: ligand / ID: 2 / Number of copies: 8 / Formula: U52
Molecular weightTheoretical: 484.978 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.48 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHepes
200.0 mMNaClSodium chlorideSodium chloride
1.0 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: LEICA EM GP
Details: 4 ul sample applied twice, blotted 1.3s each time.
DetailsStrep II-Avi BCL6 (aa5-360) polymerized by addition of 1.5 molar excess BI-3802. CHAPSO (0.8 mM final) added for grid preparation.

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Electron microscopy

MicroscopeFEI TITAN 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: -2.5 µm / Nominal defocus min: -1.0 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
DetailsData collection in counting mode, using multi shot scheme (4 holes per stage position, 2 shots per hole)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 7553 / Average exposure time: 3.0 sec. / Average electron dose: 63.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1610413 / Details: particles picked with crYOLO
Startup modelType of model: OTHER / Details: Initial model generated in Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: as implemented in Relion / Number images used: 112048
DetailsThe selected movies were corrected for beam-induced motion.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5mw2


Chain - Chain ID: A / Chain - Residue range: 7-128 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsReal_space_refine: global minimization, rigid body and adp refinement with target restraints.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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