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- EMDB-9640: 112-bp octasome/DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-9640
Title112-bp octasome/DNA complex
Map data
Sample112-bp octasome/DNA complex:
Histone H2A / Histone H2B / Histone H3.1Histone H3 / Histone H4 / 112bp 601 DNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsMayanagi K / Saikusa K / Miyazaki N / Akashi S / Iwasaki K / Nishimura Y / Morikawa K / Tsunaka Y
Funding support Japan, 11 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP17am0101073 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101072j0001 Japan
Japan Society for the Promotion of ScienceJP26251008 Japan
Japan Society for the Promotion of ScienceJP16K18528 Japan
Japan Society for the Promotion of ScienceJP16H01410 Japan
Japan Society for the Promotion of ScienceJP17K07313 Japan
Japan Society for the Promotion of Science18K06089 Japan
Japan Society for the Promotion of ScienceJP18K06064 Japan
Japan Society for the Promotion of ScienceJP26505009 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101076 Japan
Japan Science and TechnologyJPMJPR12L9 Japan
Citation
Journal: Sci Rep / Year: 2019
Title: Structural visualization of key steps in nucleosome reorganization by human FACT.
Authors: Kouta Mayanagi / Kazumi Saikusa / Naoyuki Miyazaki / Satoko Akashi / Kenji Iwasaki / Yoshifumi Nishimura / Kosuke Morikawa / Yasuo Tsunaka /
Abstract: Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry ...Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.
#1: Journal: Genes Dev / Year: 2016
Title: Integrated molecular mechanism directing nucleosome reorganization by human FACT.
Authors: Yasuo Tsunaka / Yoshie Fujiwara / Takuji Oyama / Susumu Hirose / Kosuke Morikawa /
Abstract: Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT- ...Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 29, 2018-
Header (metadata) releaseJul 31, 2019-
Map releaseJul 31, 2019-
UpdateJul 31, 2019-
Current statusJul 31, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.125
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.125
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9640.png

Downloads & links

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Map

FileDownload / File: emd_9640.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 128 pix.
= 179.2 Å		1.4 Å/pix.
x 128 pix.
= 179.2 Å		1.4 Å/pix.
x 128 pix.
= 179.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.125 / Movie #1: 0.125
Minimum - Maximum-0.3247932 - 0.49800757
Average (Standard dev.)0.005142457 (±0.034249816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 179.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z179.200179.200179.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.3250.4980.005

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Supplemental data

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Sample components

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Entire 112-bp octasome/DNA complex

EntireName: 112-bp octasome/DNA complex / Number of components: 6

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Component #1: protein, 112-bp octasome/DNA complex

ProteinName: 112-bp octasome/DNA complex / Recombinant expression: No
MassTheoretical: 200 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #2: protein, Histone H2A

ProteinName: Histone H2A / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #3: protein, Histone H2B

ProteinName: Histone H2B / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #4: protein, Histone H3.1

ProteinName: Histone H3.1Histone H3 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #5: protein, Histone H4

ProteinName: Histone H4 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #6: protein, 112bp 601 DNA

ProteinName: 112bp 601 DNA
Details: 112 bp fragment of Widom-601 strong nucleosome positioning DNA sequence
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli DH5[alpha] (bacteria) / Strain: DH5[alpha]

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL / pH: 7.5
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 20 K / Humidity: 99 %

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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: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 37441
3D reconstructionSoftware: RELION / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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