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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 dataPhase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle (class S0)
Sample
  • Complex: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
    • Complex: CENP-A
    • Complex: Histones H2A, H2B, H4
    • Complex: 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
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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)
AnnotationPhase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle (class S0)
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
Components
  • Complex: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
    • Complex: CENP-A
    • Complex: Histones H2A, H2B, H4
    • Complex: Widom 601 DNA

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Supramolecule #1: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 ...

SupramoleculeName: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6

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Supramolecule #2: CENP-A

SupramoleculeName: CENP-A / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Histones H2A, H2B, H4

SupramoleculeName: Histones H2A, H2B, H4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: Widom 601 DNA

SupramoleculeName: Widom 601 DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-38 / Number grids imaged: 2 / Number real images: 2608 / Average exposure time: 7.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.3 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 47000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 227552
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Software - details: https://www.mrc-lmb.cam.ac.uk/kzhang/
Startup modelType of model: OTHER
Details: Low-pass filtered EM map of the canonical H3 nucleosome core particle
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.03) / Number images used: 87691
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.03)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.03)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 2.03)

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