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- PDB-7ssa: Cryo-EM structure of pioneer factor Cbf1 bound to the nucleosome -

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Basic information

Entry
Database: PDB / ID: 7ssa
TitleCryo-EM structure of pioneer factor Cbf1 bound to the nucleosome
Components
  • (DNA (137-MER)) x 2
  • Centromere-binding protein 1
  • Histone H2A.1
  • Histone H2B.1
  • Histone H3.2
  • Histone H4
KeywordsGENE REGULATION / Transcription factor / basic helix-loop-helix / complex
Function / homology
Function and homology information


Cbf1-Met4-Met28 complex / positive regulation of sulfate assimilation / regulation of sulfur metabolic process / positive regulation of inositol biosynthetic process / negative regulation of ceramide biosynthetic process / HATs acetylate histones / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / centromeric DNA binding ...Cbf1-Met4-Met28 complex / positive regulation of sulfate assimilation / regulation of sulfur metabolic process / positive regulation of inositol biosynthetic process / negative regulation of ceramide biosynthetic process / HATs acetylate histones / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / centromeric DNA binding / Assembly of the ORC complex at the origin of replication / HDACs deacetylate histones / cellular response to methionine / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Oxidative Stress Induced Senescence / RMTs methylate histone arginines / DNA damage tolerance / RNA Polymerase I Promoter Escape / Estrogen-dependent gene expression / chromosome, centromeric region / Ub-specific processing proteases / chromosome segregation / kinetochore / DNA-binding transcription repressor activity, RNA polymerase II-specific / structural constituent of chromatin / nucleosome / heterochromatin formation / nucleosome assembly / chromatin organization / DNA-binding transcription activator activity, RNA polymerase II-specific / RNA polymerase II-specific DNA-binding transcription factor binding / protein dimerization activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / chromatin remodeling / DNA-binding transcription factor activity / protein heterodimerization activity / DNA repair / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / nucleoplasm / nucleus
Similarity search - Function
: / Helix-loop-helix DNA-binding domain / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Helix-loop-helix DNA-binding domain superfamily / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. ...: / Helix-loop-helix DNA-binding domain / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Helix-loop-helix DNA-binding domain superfamily / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2B.1 / Histone H2A.1 / Centromere-binding protein 1 / Histone H4 / Histone H3.2
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Saccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsEek, P. / Tan, S.
Funding support United States, Estonia, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127034 United States
Estonian Research CouncilPUTJD906 Estonia
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM121858 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM131626 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM139564 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM139654 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM086252 United States
National Science Foundation (NSF, United States)1715321 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Mol Cell / Year: 2023
Title: Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome-depleted regions.
Authors: Benjamin T Donovan / Hengye Chen / Priit Eek / Zhiyuan Meng / Caroline Jipa / Song Tan / Lu Bai / Michael G Poirier /
Abstract: Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding ...Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single-molecule fluorescence studies show that the Cbf1 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones, whereas the Pho4 HLH region does not. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single-molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by PFs and how this translates to facilitating chromatin opening inside cells.
History
DepositionNov 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 3, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A.1
D: Histone H2B.1
E: Histone H3.2
F: Histone H4
G: Histone H2A.1
H: Histone H2B.1
K: Centromere-binding protein 1
L: Centromere-binding protein 1
I: DNA (137-MER)
J: DNA (137-MER)


Theoretical massNumber of molelcules
Total (without water)280,35812
Polymers280,35812
Non-polymers00
Water00
1


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

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Components

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Protein , 5 types, 10 molecules AEBFCGDHKL

#1: Protein Histone H3.2


Mass: 15303.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a-xH3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: P84233
#2: Protein Histone H4


Mass: 11394.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a-xH4 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: P62799
#3: Protein Histone H2A.1


Mass: 13881.980 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: HTA1, H2A1, SPT11, YDR225W, YD9934.10 / Plasmid: pET11a-yH2A / Production host: Escherichia coli (E. coli) / Strain (production host): CodonPlus(DE3) / References: UniProt: P04911
#4: Protein Histone H2B.1 / Suppressor of Ty protein 12


Mass: 14149.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: HTB1, H2B1, SPT12, YDR224C, YD9934.09C / Plasmid: pET11a-yH2B / Production host: Escherichia coli (E. coli) / Strain (production host): CodonPlus(DE3) / References: UniProt: P02293
#5: Protein Centromere-binding protein 1 / CBP-1 / Centromere promoter factor 1 / Centromere-binding factor 1


Mass: 39457.648 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CBF1, CEP1, CP1, CPF1, YJR060W, J1730 / Plasmid: pST50Tr-STRaHSTNyCbf1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: P17106

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DNA chain , 2 types, 2 molecules IJ

#6: DNA chain DNA (137-MER)


Mass: 45796.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST103-16xNCP601a149c15 / Production host: Escherichia coli HB101 (bacteria)
#7: DNA chain DNA (137-MER)


Mass: 46187.418 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST103-16xNCP601a149c15 / Production host: Escherichia coli HB101 (bacteria)

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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
IDNameTypeEntity IDParent-IDSource
1Pioneer factor Cbf1 in complex with the nucleosomeCOMPLEXall0RECOMBINANT
2Histone H3.2, Histone H4COMPLEX#1-#21RECOMBINANT
3Histone H2A.1, Histone H2B.1COMPLEX#3-#41RECOMBINANT
4Centromere-binding protein 1COMPLEX#51RECOMBINANT
5DNACOMPLEX#6-#71RECOMBINANT
Molecular weightValue: 0.286 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
23Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)4932
12Xenopus laevis (African clawed frog)559292
35synthetic construct (others)32630
44Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli (E. coli)562
35Escherichia coli HB101 (bacteria)634468
44Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
275 mMsodium chlorideNaCl1
31 mMdithiothreitolDTT1
40.1 mMphenylmethylsulfonyl fluoridePMSF1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 18000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6339

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.2.0CTF correction
7PHENIX1.19.2model fitting
9cryoSPARC3.2.0initial Euler assignment
10cryoSPARC3.2.0final Euler assignment
11RELION3.1.2classification
12cryoSPARC3.2.03D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73243 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3LZ0

3lz0


Accession code: 3LZ0 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 66.52 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005113245
ELECTRON MICROSCOPYf_angle_d0.637319057
ELECTRON MICROSCOPYf_chiral_restr0.04232181
ELECTRON MICROSCOPYf_plane_restr0.00571465
ELECTRON MICROSCOPYf_dihedral_angle_d30.09833708

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