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- PDB-7bxt: The cryo-EM structure of CENP-A nucleosome in complex with CENP-C... -

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Basic information

Entry
Database: PDB / ID: 7bxt
TitleThe cryo-EM structure of CENP-A nucleosome in complex with CENP-C peptide and CENP-N N-terminal domain
Components
  • (DNA (145-mer)) x 2
  • CENP-C
  • Histone H2A type 1-B/E
  • Histone H2B type 2-E
  • Histone H3,Histone H3-like centromeric protein A
  • Histone H4
  • Maltodextrin-binding protein,Centromere protein N
KeywordsCELL CYCLE/DNA / CENP-A nucleosome / CENP-C / CENP-N / complex / kinetochore / NUCLEAR PROTEIN / CELL CYCLE-DNA complex
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 / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates 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 / Metalloprotease DUBs / kinetochore / 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 / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / 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 / RUNX1 regulates transcription of genes involved in differentiation of HSCs / outer membrane-bounded periplasmic space / chromatin organization / Processing of DNA double-strand break ends / HATs acetylate histones / 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 / RmlC-like jelly roll fold / 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 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
DNA / DNA (> 10) / DNA (> 100) / 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 ...DNA / DNA (> 10) / DNA (> 100) / 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)
Homo sapiens (human)
unidentified cloning vector (others)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAriyoshi, M. / Makino, F. / Fukagawa, T.
Funding support Japan, 3items
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 20, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 10, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • EMDB-30237
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Assembly

Deposited unit
A: Histone H3,Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 2-E
E: Histone H3,Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 2-E
I: DNA (145-mer)
J: DNA (145-mer)
K: CENP-C
L: CENP-C
M: Maltodextrin-binding protein,Centromere protein N
N: Maltodextrin-binding protein,Centromere protein N


Theoretical massNumber of molelcules
Total (without water)355,87814
Polymers355,87814
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area65190 Å2
ΔGint-433 kcal/mol
Surface area93110 Å2

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Components

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Protein , 5 types, 10 molecules AEBFCGDHMN

#1: Protein Histone H3,Histone H3-like centromeric protein A / Centromere protein A / GgCENP-A


Mass: 16372.217 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: chimeric protein, chicken histone H3 (aa 1-64)-chicken CENP-A (aa 52-131)
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: pET15b / Gene: CENPA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q6XXM1, UniProt: P84229*PLUS
#2: Protein Histone H4 /


Mass: 11676.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein Histone H2B type 2-E / H2B-clustered histone 21 / Histone H2B-GL105 / Histone H2B.q / H2B/q


Mass: 14233.518 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC21, H2BFQ, HIST2H2BE / Production host: Escherichia coli (E. coli) / References: UniProt: Q16778
#8: Protein Maltodextrin-binding protein,Centromere protein N / CENP-N


Mass: 71519.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The fusion protein of N-terminal MBP, N-terminal domain of chicken CENP-N and C-terminal histidine tags
Source: (gene. exp.) unidentified cloning vector (others), (gene. exp.) Gallus gallus (chicken)
Gene: CENPN / Details (production host): pMAL_c2X / Production host: Escherichia coli (E. coli) / References: UniProt: Q1T765, UniProt: P0AEX9*PLUS

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-mer)


Mass: 44529.398 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (145-mer)


Mass: 44982.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein/peptide , 1 types, 2 molecules KL

#7: Protein/peptide CENP-C / Centromere protein CENP-C


Mass: 4932.840 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Gallus gallus (chicken) / References: UniProt: O57392, UniProt: F1NSD9*PLUS

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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 in complex with CENP-C peptide and CNEP-N N-terminal domainCOMPLEXall0MULTIPLE SOURCES
2Histone H3,Histone H3-like centromeric protein ACOMPLEX#11MULTIPLE SOURCES
3Histone H4COMPLEX#21MULTIPLE SOURCES
4Histone H2A type 1-B/ECOMPLEX#31MULTIPLE SOURCES
5Histone H2B type 2-ECOMPLEX#41MULTIPLE SOURCES
6DNACOMPLEX#5-#61MULTIPLE SOURCES
7CENP-CCOMPLEX#71MULTIPLE SOURCES
8Maltodextrin-binding protein,Centromere protein NCOMPLEX#81MULTIPLE SOURCES
Molecular weightValue: 0.33 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Ectocarpus siliculosus (eukaryote)2880
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
85Escherichia coli (strain B / BL21-DE3) (bacteria)469008
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaClSodium chloride1
32 mMDTTC4H10O2S21
41 mMEDTAEthylenediaminetetraacetic acidC10H16N2O81
50.1 %CHAPSC32H58N2O7S1
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Both side / Grid material: MOLYBDENUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 45454 X / Nominal defocus max: -3500 nm / Nominal defocus min: -1000 nm / Calibrated defocus min: -500 nm / Calibrated defocus max: -7000 nm / Cs: 1.4 mm / C2 aperture diameter: 150 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 1.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 5346
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
1RELION3.08particle selection
4GctfCTF correction
10RELION3.08initial Euler assignment
11RELION3.08final Euler assignment
12RELION3.08classification
13RELION3.083D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 421635
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118294 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE

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