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- EMDB-30241: The cryo-EM density map of CENP-A nucleosome in complex with asym... -

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Basic information

Entry
Database: EMDB / ID: EMD-30241
TitleThe cryo-EM density map of CENP-A nucleosome in complex with asymmetric CENP-C C-terminal domain and CENP-N N-terminal domain
Map dataThe cryo-EM density map of CENP-A nucleosome in complex with CENP-C C-terminal domain and CENP-N N-terminal domain
Sample
  • Complex: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
Function / homology
Function and homology information


Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / Resolution of Sister Chromatid Cohesion / HATs acetylate histones / EML4 and NUDC in mitotic spindle formation / kinetochore => GO:0000776 / RHO GTPases Activate Formins / PRC2 methylates histones and DNA / Separation of Sister Chromatids ...Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / Resolution of Sister Chromatid Cohesion / HATs acetylate histones / EML4 and NUDC in mitotic spindle formation / kinetochore => GO:0000776 / RHO GTPases Activate Formins / PRC2 methylates histones and DNA / Separation of Sister Chromatids / Oxidative Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / Assembly of the ORC complex at the origin of replication / RNA Polymerase I Promoter Opening / RNA Polymerase I Promoter Escape / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Estrogen-dependent gene expression / Factors involved in megakaryocyte development and platelet production / spindle attachment to meiosis I kinetochore / centromeric DNA binding / CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / condensed chromosome, centromeric region / detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / carbohydrate transport / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / telomere organization / RNA Polymerase I Promoter Opening / ATP-binding cassette (ABC) transporter complex / SUMOylation of chromatin organization proteins / Assembly of the ORC complex at the origin of replication / cell chemotaxis / DNA methylation / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / innate immune response in mucosa / PRC2 methylates histones and DNA / Defective pyroptosis / chromosome segregation / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / G2/M DNA damage checkpoint / B-WICH complex positively regulates rRNA expression / HDMs demethylate histones / DNA Damage/Telomere Stress Induced Senescence / kinetochore / Metalloprotease DUBs / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / structural constituent of chromatin / Transcriptional regulation of granulopoiesis / UCH proteinases / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / outer membrane-bounded periplasmic space / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / HATs acetylate histones / Processing of DNA double-strand break ends / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence
Similarity search - Function
Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Mif2/CENP-C cupin domain / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C like / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. ...Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Mif2/CENP-C cupin domain / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C like / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / RmlC-like cupin domain superfamily / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / 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 / RmlC-like jelly roll fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / 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
Uncharacterized protein / CENP-C / Histone H2A type 1-B/E / Maltose/maltodextrin-binding periplasmic protein / Histone H4 / Histone H3.2 / Histone H2B type 2-E / Centromere protein N / Histone H3-like centromeric protein A
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsAriyoshi M / Makino F
Funding support Japan, 3 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)25221106 Japan
Japan Agency for Medical Research and Development (AMED)0101117 Japan
Japan Agency for Medical Research and Development (AMED)0101020 Japan
CitationJournal: EMBO J / Year: 2021
Title: Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.
Authors: Mariko Ariyoshi / Fumiaki Makino / Reito Watanabe / Reiko Nakagawa / Takayuki Kato / Keiichi Namba / Yasuhiro Arimura / Risa Fujita / Hitoshi Kurumizaka / Ei-Ichi Okumura / Masatoshi Hara / Tatsuo Fukagawa /
Abstract: The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a ...The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.
History
DepositionApr 21, 2020-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateMar 10, 2021-
Current statusMar 10, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_30241.map.gz / Format: CCP4 / Size: 36.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe cryo-EM density map of CENP-A nucleosome in complex with CENP-C C-terminal domain and CENP-N N-terminal domain
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.013991567 - 0.056325626
Average (Standard dev.)0.0010497316 (±0.0042412444)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions212212212
Spacing212212212
CellA=B=C: 233.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z212212212
origin x/y/z0.0000.0000.000
length x/y/z233.200233.200233.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ296296296
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS212212212
D min/max/mean-0.0140.0560.001

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Supplemental data

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Sample components

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Entire : CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-ter...

EntireName: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
Components
  • Complex: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain

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Supramolecule #1: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-ter...

SupramoleculeName: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 330 KDa

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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

Concentration1.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chloridesodium chloride
2.0 mMC4H10O2S2DTT
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
0.1 %C32H58N2O7SCHAPS
GridModel: Quantifoil R0.6/1 / Material: MOLYBDENUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 300.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 200
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Calibrated defocus max: 7.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 45454 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.4 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 5 / Number real images: 5346 / Average exposure time: 10.0 sec. / Average electron dose: 1.2 e/Å2

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Image processing

Particle selectionNumber selected: 343497
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.08)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 3959
FSC plot (resolution estimation)

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