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- EMDB-25406: Cryo-EM structure of pioneer factor Cbf1 bound to the nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-25406
TitleCryo-EM structure of pioneer factor Cbf1 bound to the nucleosome
Map datamain map from cryoSPARC refinement
Sample
  • Complex: Pioneer factor Cbf1 in complex with the nucleosome
    • Complex: Histone H3.2, Histone H4
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
    • Complex: Histone H2A.1, Histone H2B.1
      • Protein or peptide: Histone H2A.1
      • Protein or peptide: Histone H2B.1
    • Complex: Centromere-binding protein 1
      • Protein or peptide: Centromere-binding protein 1
    • Complex: DNA
      • DNA: DNA (137-MER)
      • DNA: DNA (137-MER)
Function / homology
Function and homology information


Cbf1-Met4-Met28 complex / regulation of sulfur metabolic process / Condensation of Prophase Chromosomes / centromeric DNA binding / replication fork protection complex / RMTs methylate histone arginines / postreplication repair / chromosome, centromeric region / chromosome segregation / kinetochore ...Cbf1-Met4-Met28 complex / regulation of sulfur metabolic process / Condensation of Prophase Chromosomes / centromeric DNA binding / replication fork protection complex / RMTs methylate histone arginines / postreplication repair / chromosome, centromeric region / chromosome segregation / kinetochore / DNA-binding transcription repressor activity, RNA polymerase II-specific / structural constituent of chromatin / nucleosome / chromatin organization / DNA-binding transcription activator activity, RNA polymerase II-specific / RNA polymerase II-specific DNA-binding transcription factor binding / protein dimerization activity / chromatin remodeling / RNA polymerase II cis-regulatory region sequence-specific DNA binding / 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 DNA-binding domain superfamily / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...: / Helix-loop-helix DNA-binding domain / Helix-loop-helix DNA-binding domain superfamily / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
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 (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsEek P / Tan S
Funding support United States, Estonia, 9 items
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, 2021-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25406.png

Downloads & links

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Map

FileDownload / File: emd_25406.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map from cryoSPARC refinement
Voxel sizeX=Y=Z: 1.288 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum0.0 - 1.0657156
Average (Standard dev.)0.006083665 (±0.02645681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 329.728 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25406_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_25406_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: map postprocessed with deepEMhancer

Fileemd_25406_additional_1.map
Annotationmap postprocessed with deepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A from cryoSPARC refinement

Fileemd_25406_half_map_1.map
Annotationhalf map A from cryoSPARC refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B from cryoSPARC refinement

Fileemd_25406_half_map_2.map
Annotationhalf map B from cryoSPARC 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 : Pioneer factor Cbf1 in complex with the nucleosome

EntireName: Pioneer factor Cbf1 in complex with the nucleosome
Components
  • Complex: Pioneer factor Cbf1 in complex with the nucleosome
    • Complex: Histone H3.2, Histone H4
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
    • Complex: Histone H2A.1, Histone H2B.1
      • Protein or peptide: Histone H2A.1
      • Protein or peptide: Histone H2B.1
    • Complex: Centromere-binding protein 1
      • Protein or peptide: Centromere-binding protein 1
    • Complex: DNA
      • DNA: DNA (137-MER)
      • DNA: DNA (137-MER)

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Supramolecule #1: Pioneer factor Cbf1 in complex with the nucleosome

SupramoleculeName: Pioneer factor Cbf1 in complex with the nucleosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 286 KDa

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Supramolecule #2: Histone H3.2, Histone H4

SupramoleculeName: Histone H3.2, Histone H4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Supramolecule #3: Histone H2A.1, Histone H2B.1

SupramoleculeName: Histone H2A.1, Histone H2B.1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

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Supramolecule #4: Centromere-binding protein 1

SupramoleculeName: Centromere-binding protein 1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

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Supramolecule #5: DNA

SupramoleculeName: DNA / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #6-#7
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 15.30393 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAV MALQEASEAY LVALFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 11.394426 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVY ALKRQGRTLY GFGG

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Macromolecule #3: Histone H2A.1

MacromoleculeName: Histone H2A.1 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 13.88198 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGGKGGKAGS AAKASQSRSA KAGLTFPVGR VHRLLRRGNY AQRIGSGAPV YLTAVLEYLA AEILELAGNA ARDNKKTRII PRHLQLAIR NDDELNKLLG NVTIAQGGVL PNIHQNLLPK KSAKATKASQ EL

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Macromolecule #4: Histone H2B.1

MacromoleculeName: Histone H2B.1 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 14.149168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SAKAEKKPAS KAPAEKKPAA KKTSTSTDGK KRSKARKETY SSYIYKVLKQ THPDTGISQK SMSILNSFVN DIFERIATEA SKLAAYNKK STISAREIQT AVRLILPGEL AKHAVSEGTR AVTKYSSSTQ A

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Macromolecule #5: Centromere-binding protein 1

MacromoleculeName: Centromere-binding protein 1 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 39.457648 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSNSLANNNK LSTEDEEIHS ARKRGYNEEQ NYSEARKKQR DQGLLSQESN DGNIDSALLS EGATLKGTQS QYESGLTSNK DEKGSDDED ASVAEAAVAA TVNYTDLIQG QEDSSDAHTS NQTNANGEHK DSLNGERAIT PSNEGVKPNT SLEGMTSSPM E STQQSKND ...String:
GSNSLANNNK LSTEDEEIHS ARKRGYNEEQ NYSEARKKQR DQGLLSQESN DGNIDSALLS EGATLKGTQS QYESGLTSNK DEKGSDDED ASVAEAAVAA TVNYTDLIQG QEDSSDAHTS NQTNANGEHK DSLNGERAIT PSNEGVKPNT SLEGMTSSPM E STQQSKND MLIPLAEHDR GPEHQQDDED NDDADIDLKK DISMQPGRRG RKPTTLATTD EWKKQRKDSH KEVERRRREN IN TAINVLS DLLPVRESSK AAILACAAEY IQKLKETDEA NIEKWTLQKL LSEQNASQLA SANEKLQEEL GNAYKEIEYM KRV LRKEGI EYEDMHTHKK QENERKSTRS DNPHEA

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Macromolecule #6: DNA (137-MER)

MacromoleculeName: DNA (137-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 45.796195 KDa
SequenceString: (DA)(DT)(DC)(DG)(DG)(DA)(DG)(DA)(DG)(DG) (DT)(DC)(DA)(DC)(DG)(DT)(DG)(DA)(DC)(DC) (DA)(DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC) (DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT) (DA) (DG)(DA)(DC)(DA)(DG)(DC) ...String:
(DA)(DT)(DC)(DG)(DG)(DA)(DG)(DA)(DG)(DG) (DT)(DC)(DA)(DC)(DG)(DT)(DG)(DA)(DC)(DC) (DA)(DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC) (DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT) (DA) (DG)(DA)(DC)(DA)(DG)(DC)(DT)(DC) (DT)(DA)(DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT) (DT)(DA) (DA)(DA)(DC)(DG)(DC)(DA)(DC) (DG)(DT)(DA)(DC)(DG)(DC)(DG)(DC)(DT)(DG) (DT)(DC)(DC) (DC)(DC)(DC)(DG)(DC)(DG) (DT)(DT)(DT)(DT)(DA)(DA)(DC)(DC)(DG)(DC) (DC)(DA)(DA)(DG) (DG)(DG)(DG)(DA)(DT) (DT)(DA)(DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG) (DT)(DC)(DT)(DC)(DC) (DA)(DG)(DG)(DG) (DA)(DC)(DG)(DT)(DC)(DT)(DC)(DA)(DG)(DA) (DT)(DA)(DT)(DA)(DT)(DA) (DC)(DA)(DT) (DC)(DC)(DT)(DG)(DA)(DT)

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Macromolecule #7: DNA (137-MER)

MacromoleculeName: DNA (137-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 46.187418 KDa
SequenceString: (DA)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DG)(DA)(DC)(DG)(DT)(DC)(DC)(DC)(DT) (DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG) (DG) (DA)(DG)(DT)(DA)(DA)(DT) ...String:
(DA)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DG)(DA)(DC)(DG)(DT)(DC)(DC)(DC)(DT) (DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG) (DG) (DA)(DG)(DT)(DA)(DA)(DT)(DC)(DC) (DC)(DC)(DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT) (DT)(DA) (DA)(DA)(DA)(DC)(DG)(DC)(DG) (DG)(DG)(DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG) (DC)(DG)(DT) (DA)(DC)(DG)(DT)(DG)(DC) (DG)(DT)(DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG) (DT)(DG)(DC)(DT) (DA)(DG)(DA)(DG)(DC) (DT)(DG)(DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC) (DC)(DA)(DA)(DT)(DT) (DG)(DA)(DG)(DC) (DG)(DG)(DC)(DC)(DT)(DG)(DG)(DT)(DC)(DA) (DC)(DG)(DT)(DG)(DA)(DC) (DC)(DT)(DC) (DT)(DC)(DC)(DG)(DA)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
75.0 mMNaClSodium chloridesodium chloride
1.0 mMDTTdithiothreitol
0.1 mMPMSFphenylmethylsulfonyl fluoridePMSF
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 18000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6339 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.2)
Details: Final classification performed without alignment using a mask containing only the volume of Cbf1
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 73243
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3lz0

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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