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- EMDB-8140: 3.87 Angstrom structure of the nucleosome core particle obtained ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8140
Title3.87 Angstrom structure of the nucleosome core particle obtained by phase-plate cryo-EM
Map data
SampleNucleosome core particle with 145 base pairs of the Widom 601 sequence.:
Widom 601 DNA, 145 base pairs / Histone octamer / nucleic-acidNucleic acid / Histone 2A / Histone 2B / Histone 3 / Histone 4
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsChua EYD / Vogirala VK / Inian O / Wong ASW / Plitzko JM / Danev R / Sandin S
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education Singapore
CitationJournal: Nucleic Acids Res / Year: 2016
Title: 3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM.
Authors: Eugene Y D Chua / Vinod K Vogirala / Oviya Inian / Andrew S W Wong / Lars Nordenskiöld / Juergen M Plitzko / Radostin Danev / Sara Sandin /
Abstract: The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining ...The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMar 31, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseSep 7, 2016-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8140.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 160 pix.
= 220.8 Å
1.38 Å/pix.
x 160 pix.
= 220.8 Å
1.38 Å/pix.
x 160 pix.
= 220.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.056169275 - 0.11585718
Average (Standard dev.)0.00018258722 (±0.0066085593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0560.1160.000

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Supplemental data

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Sample components

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Entire Nucleosome core particle with 145 base pairs of the Widom 601 seq...

EntireName: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
Number of components: 8

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Component #1: protein, Nucleosome core particle with 145 base pairs of the Wido...

ProteinName: Nucleosome core particle with 145 base pairs of the Widom 601 sequence.
Recombinant expression: No
MassExperimental: 200 kDa
SourceSpecies: Xenopus laevis (African clawed frog)

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Component #2: protein, Widom 601 DNA, 145 base pairs

ProteinName: Widom 601 DNA, 145 base pairs / Recombinant expression: No
MassExperimental: 90 kDa
SourceSpecies: synthetic construct (others)

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Component #3: protein, Histone octamer

ProteinName: Histone octamer / Recombinant expression: No
MassExperimental: 110 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pUC57

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Component #4: nucleic-acid, Widom 601 DNA, 145 base pairs

nucleic acidName: Widom 601 DNA, 145 base pairs / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
ATCAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCGAT
SourceSpecies: synthetic construct (others)

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Component #5: protein, Histone 2A

ProteinName: Histone 2A / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Histone 2B

ProteinName: Histone 2B / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, Histone 3

ProteinName: Histone 3 / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: protein, Histone 4

ProteinName: Histone 4 / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
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 solutionSpecimen conc.: 2.5 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Details: 3-4 seconds blotting time.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 31 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 26060
Details: The recorded micrographs were clipped to remove dust and the movie frames were motion-corrected.
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Details: RELION 1.4 was used for the final reconstruction.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 3LZ0
Overall bvalue: 78.96

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