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- PDB-9b9r: Cryo-EM structure of the ZBTB5 BTB domain filament -

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Basic information

Entry
Database: PDB / ID: 9b9r
TitleCryo-EM structure of the ZBTB5 BTB domain filament
ComponentsZinc finger and BTB domain-containing protein 5
KeywordsTRANSCRIPTION / BTB domain / transcription factor / ZBTB protein
Function / homology
Function and homology information


RNA polymerase II transcription regulatory region sequence-specific DNA binding / DNA-binding transcription repressor activity, RNA polymerase II-specific / DNA-binding transcription factor activity, RNA polymerase II-specific / chromatin / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / nucleus / metal ion binding
Similarity search - Function
BTB/POZ domain / BTB domain profile. / 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
Zinc finger and BTB domain-containing protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPark, J. / Hunkeler, M. / Fischer, E.S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2024
Title: Polymerization of ZBTB transcription factors regulates chromatin occupancy.
Authors: Paul M C Park / Jiho Park / Jared Brown / Moritz Hunkeler / Shourya S Roy Burman / Katherine A Donovan / Hojong Yoon / Radosław P Nowak / Mikołaj Słabicki / Benjamin L Ebert / Eric S Fischer /
Abstract: BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, ...BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.
History
DepositionApr 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Zinc finger and BTB domain-containing protein 5
B: Zinc finger and BTB domain-containing protein 5
C: Zinc finger and BTB domain-containing protein 5
D: Zinc finger and BTB domain-containing protein 5


Theoretical massNumber of molelcules
Total (without water)75,1454
Polymers75,1454
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Zinc finger and BTB domain-containing protein 5


Mass: 18786.326 Da / Num. of mol.: 4 / Fragment: BTB domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZBTB5, KIAA0354 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O15062

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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: ZBTB5 BTB domain filament / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: High Five
Buffer solutionpH: 7.4
Details: 50 mM HEPES/NaOH pH 7.4, 200 mM NaCl, 1 mM CHAPSO, 1 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES/NaOH pH 7.4C8H18N2O4S/NaOH1
2200 mMNaClNaCl1
31 mMCHAPSOC32H58N2O8S1
41 mMTCEPC9H15O6P1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Elution fractions from Strep-tag affinity chromatography were dialyzed overnight against 50 mM HEPES/NaOH pH 7.4, 200 mM NaCl, 1 mM TCEP and concentrated by centrifugation.
Specimen supportDetails: Grids were glow-discharged for 60 s at 15-20 mA and 39 Pa.
Grid material: COPPER / 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: Grids were vitrified using a Leica EM GP plunge freezer operated at 90% humidity and 10 C with 10 s pre-blot, 3 s blot, 3 s post-blot.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 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: 2.1 sec. / Electron dose: 50.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10854
Details: 2 movies (46 frames) were acquired per hole with 9 holes per stage position.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.1particle selectionTemplate-based picking
2SerialEM4.1bimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fitting
10cryoSPARC4.4.1initial Euler assignment
11cryoSPARC4.4.1final Euler assignment
13cryoSPARC4.4.13D reconstruction
14PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6344567
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1352367 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 194 / Protocol: OTHER / Space: REAL / Details: Real-space refinement without local grid search
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 206.63 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00223560
ELECTRON MICROSCOPYf_angle_d0.67634808
ELECTRON MICROSCOPYf_chiral_restr0.0393560
ELECTRON MICROSCOPYf_plane_restr0.0048608
ELECTRON MICROSCOPYf_dihedral_angle_d11.04161244

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