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- EMDB-10822: CENP-A nucleosome core particle with 145 base pairs of the Widom ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10822
TitleCENP-A nucleosome core particle with 145 base pairs of the Widom 601 sequence
Map dataPhase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle
Sample
  • Complex: CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequence
    • Complex: CENP-ACENPA
    • Complex: Histones H2A, H2B, H4
    • Complex: Widom 601 DNA
Function / homology
Function and homology information


CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / protein localization to CENP-A containing chromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / pericentric heterochromatin ...CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / protein localization to CENP-A containing chromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / pericentric heterochromatin / CENP-A containing nucleosome / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / RHO GTPases Activate Formins / structural constituent of chromatin / Separation of Sister Chromatids / nucleosome / protein heterodimerization activity / chromatin binding / DNA binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site ...Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
Histone H2B 1.1 / Histone H2A type 1 / Histone H3-like centromeric protein A / Histone H4 / Histone H2A
Similarity search - Component
Biological speciesHomo sapiens (human) / Xenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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 3, 2020-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 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: 0.0735
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0735
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tem
  • Surface level: 0.0735
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10822.png

Downloads & links

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Map

FileDownload / File: emd_10822.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
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0735 / Movie #1: 0.0735
Minimum - Maximum-0.33032772 - 0.47063097
Average (Standard dev.)0.0024075313 (±0.028323965)
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-0.3300.4710.002

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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-ACENPA
    • 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 / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 47000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: 0.5 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
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
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.03)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 2.03)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.03)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.03) / Number images used: 63968

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 38.2 / Target criteria: Correlation coefficient
Output model

PDB-6tem:
CENP-A nucleosome core particle with 145 base pairs of the Widom 601 sequence by cryo-EM

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