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- EMDB-10823: Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10823
TitlePhase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle (class S0)
Map data
SampleCENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence:
CENP-ACENPA / Histones H2A, H2B, H4 / Widom 601 DNA
Biological speciesHomo sapiens (human) / Xenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBoopathi R / Danev R / Khoshouei M / Kale S / Nahata S / Ramos L / Angelov D / Dimitrov S / Hamiche A / Petosa C / Bednar J
Funding support France, 4 items
OrganizationGrant numberCountry
French National Research AgencyChromcomp France
French National Research AgencyChrom3D France
French National Research AgencyChrome France
French National Research AgencyEPIVAR.Z France
CitationJournal: Nucleic Acids Res. / Year: 2020
Title: Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends.
Authors: Ramachandran Boopathi / Radostin Danev / Maryam Khoshouei / Seyit Kale / Sunil Nahata / Lorrie Ramos / Dimitar Angelov / Stefan Dimitrov / Ali Hamiche / Carlo Petosa / Jan Bednar /
Abstract: The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with ...The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
History
DepositionApr 4, 2020-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateJun 10, 2020-
Current statusJun 10, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0228
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0228
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10823.png

Downloads & links

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Map

FileDownload / File: emd_10823.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 140 pix.
= 148.4 Å		1.06 Å/pix.
x 140 pix.
= 148.4 Å		1.06 Å/pix.
x 140 pix.
= 148.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0228 / Movie #1: 0.0228
Minimum - Maximum-0.07858377 - 0.12424013
Average (Standard dev.)0.0011154716 (±0.0085943695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 148.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z148.400148.400148.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0790.1240.001

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Supplemental data

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Sample components

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Entire CENP-A Nucleosome core particle with 145 base pairs of Widom 601 ...

EntireName: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
Number of components: 4

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Component #1: protein, CENP-A Nucleosome core particle with 145 base pairs of W...

ProteinName: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
Recombinant expression: No

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Component #2: protein, CENP-A

ProteinName: CENP-ACENPA / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Histones H2A, H2B, H4

ProteinName: Histones H2A, H2B, H4 / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Widom 601 DNA

ProteinName: Widom 601 DNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
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: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: OTHER / Temperature: 277 K / Humidity: 95 %

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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: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 47000 X (nominal), 47000 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2608

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 87691
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF

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