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- PDB-6tem: CENP-A nucleosome core particle with 145 base pairs of the Widom ... -

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Basic information

Entry
Database: PDB / ID: 6tem
TitleCENP-A nucleosome core particle with 145 base pairs of the Widom 601 sequence by cryo-EM
Components
  • (Widom 601 DNA (145-MER, ...) x 2
  • Histone H2A
  • Histone H2B 1.1
  • Histone H3-like centromeric protein A
  • Histone H4
KeywordsDNA BINDING PROTEIN / CENP-A nucleosome / protein-DNA complex
Function / homology
Function and homology information


inner kinetochore / kinetochore assembly / condensed chromosome, centromeric region / protein localization to chromosome, centromeric region / kinetochore => GO:0000776 / establishment of mitotic spindle orientation / CENP-A containing chromatin assembly / chromosome, centromeric region / Deposition of new CENPA-containing nucleosomes at the centromere / mitotic cytokinesis ...inner kinetochore / kinetochore assembly / condensed chromosome, centromeric region / protein localization to chromosome, centromeric region / kinetochore => GO:0000776 / establishment of mitotic spindle orientation / CENP-A containing chromatin assembly / chromosome, centromeric region / Deposition of new CENPA-containing nucleosomes at the centromere / mitotic cytokinesis / pericentric heterochromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / RHO GTPases Activate Formins / DNA-templated transcription, initiation / mitotic spindle organization / nucleosome / Separation of Sister Chromatids / protein heterodimerization activity / chromatin binding / viral process / DNA binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. / Histone H2A conserved site / C-terminus of histone H2A / Histone H2A, C-terminal domain / Histone H2A ...Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. / Histone H2A conserved site / C-terminus of histone H2A / Histone H2A, C-terminal domain / Histone H2A / Histone 2A / Histone H4 signature. / Histone H4, conserved site / Histone H4 / Histone H4 / Centromere kinetochore component CENP-T histone fold / CENP-T/Histone H4, histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF) / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA (> 100) / DNA (> 10) / DNA / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBoopathi, R. / Danev, R. / Petosa, C. / Bednar, J.
Funding support France, 4items
OrganizationGrant numberCountry
French National Research AgencyChromcomp France
French National Research AgencyChrome France
French National Research AgencyChrom3D 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
DepositionNov 12, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2020Group: Database references / Category: pdbx_database_related
Revision 1.2Apr 22, 2020Group: Database references / Category: database_2 / pdbx_database_related / Item: _database_2.database_code
Revision 1.3Apr 29, 2020Group: Database references / Category: citation
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4Jun 10, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A
D: Histone H2B 1.1
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A
H: Histone H2B 1.1
I: Widom 601 DNA (145-MER, sense)
J: Widom 601 DNA (145-MER, antisense)


Theoretical massNumber of molelcules
Total (without water)199,97510
Polymers199,97510
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area51760 Å2
ΔGint-376 kcal/mol
Surface area68470 Å2
MethodPISA

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3-like centromeric protein A / Centromere autoantigen A / Centromere protein A / CENP-A


Mass: 16023.630 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPA / Production host: Escherichia coli (E. coli) / References: UniProt: P49450
#2: Protein Histone H4 /


Mass: 11394.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P62799
#3: Protein Histone H2A /


Mass: 13978.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, LOC494591 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13834.070 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281

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Widom 601 DNA (145-MER, ... , 2 types, 2 molecules IJ

#5: DNA chain Widom 601 DNA (145-MER, sense)


Mass: 44552.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain Widom 601 DNA (145-MER, antisense)


Mass: 44961.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequenceCOMPLEX#1-#60RECOMBINANT
2CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequenceCOMPLEX#11RECOMBINANT
3CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequenceCOMPLEX#2-#41RECOMBINANT
4CENP-A Nucleosome core particle with 145 base pairs of Widom 601 sequenceCOMPLEX#5-#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Xenopus laevis (African clawed frog)8355
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 47000 X / Calibrated magnification: 47000 X / Nominal defocus min: 500 nm / Calibrated defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2608
EM imaging opticsPhase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 38 / Used frames/image: 2-38

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.51particle selectionhttps://www.mrc-lmb.cam.ac.uk/kzhang/
2SerialEM3.5image acquisition
4Gctf1.06CTF correctionhttps://www.mrc-lmb.cam.ac.uk/kzhang/
7Cootccp4-7.0model fitting
9RELION2.03initial Euler assignment
10RELION2.03final Euler assignment
11RELION2.03classification
12RELION2.033D reconstruction
13PHENIX1..12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 227552
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63968 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 38.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

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