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- EMDB-30070: Nucleosome with LIN28B distal enhancer DNA sequence -

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Basic information

Entry
Database: EMDB / ID: EMD-30070
TitleNucleosome with LIN28B distal enhancer DNA sequence
Map data
SampleNucleosome with LIN28B distal enhancer DNA sequence
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsEchigoya K / Koyama M / Takizawa Y / Kurumizaka H
Funding support Japan, 6 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP17H01408 Japan
Japan Society for the Promotion of Science (JSPS)JP18K14677 Japan
Japan Society for the Promotion of Science (JSPS)JP18H05534 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Society for the Promotion of Science (JSPS)JP19K06522 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101076 Japan
CitationJournal: Sci Rep / Year: 2020
Title: Nucleosome binding by the pioneer transcription factor OCT4.
Authors: Kenta Echigoya / Masako Koyama / Lumi Negishi / Yoshimasa Takizawa / Yuka Mizukami / Hideki Shimabayashi / Akari Kuroda / Hitoshi Kurumizaka /
Abstract: Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors ...Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMar 2, 2020-
Header (metadata) releaseSep 2, 2020-
Map releaseSep 2, 2020-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.75
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_30070.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 150 pix.
= 198. Å
1.32 Å/pix.
x 150 pix.
= 198. Å
1.32 Å/pix.
x 150 pix.
= 198. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 4.75 / Movie #1: 4.75
Minimum - Maximum-12.866925 - 22.983694
Average (Standard dev.)0.00000000135 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 198.00002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z198.000198.000198.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-12.86722.9840.000

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Supplemental data

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Sample components

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Entire Nucleosome with LIN28B distal enhancer DNA sequence

EntireName: Nucleosome with LIN28B distal enhancer DNA sequence / Number of components: 1

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Component #1: protein, Nucleosome with LIN28B distal enhancer DNA sequence

ProteinName: Nucleosome with LIN28B distal enhancer DNA sequence / Recombinant expression: No
MassTheoretical: 200 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 150721
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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