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- PDB-9b9v: Cryo-EM structure of the ZBTB9 BTB domain filament -

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Basic information

Entry
Database: PDB / ID: 9b9v
TitleCryo-EM structure of the ZBTB9 BTB domain filament
ComponentsZinc finger and BTB domain-containing protein 9
KeywordsTRANSCRIPTION / BTB domain / transcription factor / ZBTB protein
Function / homology
Function and homology information


DNA-binding transcription factor activity, RNA polymerase II-specific / RNA polymerase II cis-regulatory region sequence-specific DNA binding / regulation of transcription by RNA polymerase II / identical protein binding / 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 9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.1 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 9
B: Zinc finger and BTB domain-containing protein 9
C: Zinc finger and BTB domain-containing protein 9
D: Zinc finger and BTB domain-containing protein 9
E: Zinc finger and BTB domain-containing protein 9
F: Zinc finger and BTB domain-containing protein 9
G: Zinc finger and BTB domain-containing protein 9
H: Zinc finger and BTB domain-containing protein 9
I: Zinc finger and BTB domain-containing protein 9
J: Zinc finger and BTB domain-containing protein 9
K: Zinc finger and BTB domain-containing protein 9
L: Zinc finger and BTB domain-containing protein 9
M: Zinc finger and BTB domain-containing protein 9
N: Zinc finger and BTB domain-containing protein 9


Theoretical massNumber of molelcules
Total (without water)271,43114
Polymers271,43114
Non-polymers00
Water00
1


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

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Components

#1: Protein
Zinc finger and BTB domain-containing protein 9


Mass: 19387.961 Da / Num. of mol.: 14 / Fragment: BTB domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZBTB9 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96C00

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: ZBTB9 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, 0.25 mM CHAPSO, 1 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES/NaOH pH 7.4C8H18N2O4S/NaOH1
2200 mMNaClNaCl1
30.25 mMCHAPSOC32H58N2O8S1
41 mMTCEPC9H15O6P1
SpecimenConc.: 0.95 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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
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: 4.5 sec. / Electron dose: 50.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1137
Details: 1 movie (45 frames) was acquired per hole and stage position.

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 101.9 ° / Axial rise/subunit: 36.1 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 2022676
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147135 / Algorithm: FOURIER SPACE
Details: Asymmetric helical refinement followed by local refinement led to the final reconstruction. The helical symmetry values represent the angular rotation and axial rise for two dimers at the ...Details: Asymmetric helical refinement followed by local refinement led to the final reconstruction. The helical symmetry values represent the angular rotation and axial rise for two dimers at the core of the map, where the local resolution was the highest. These values, however, were not applied during helical refinement as they did not improve the quality of the final reconstruction.
Symmetry type: HELICAL
Atomic model buildingB value: 762 / 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: 177.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002913664
ELECTRON MICROSCOPYf_angle_d0.650418536
ELECTRON MICROSCOPYf_chiral_restr0.03912184
ELECTRON MICROSCOPYf_plane_restr0.0052380
ELECTRON MICROSCOPYf_dihedral_angle_d10.95985096

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