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- PDB-7by0: The cryo-EM structure of CENP-A nucleosome in complex with the ph... -
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Basic information
Entry | Database: PDB / ID: 7by0 | ||||||||||||
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Title | The cryo-EM structure of CENP-A nucleosome in complex with the phosphorylated CENP-C | ||||||||||||
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![]() | CELL CYCLE / CENP-A nucleosome / CENP-C / CENP-N / complex / kinetochore / NUCLEAR PROTEIN | ||||||||||||
Function / homology | ![]() Interleukin-7 signaling / Chromatin modifying enzymes / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / HDACs deacetylate histones / HATs acetylate histones / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 ...Interleukin-7 signaling / Chromatin modifying enzymes / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / HDACs deacetylate histones / HATs acetylate histones / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / 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 / centromeric DNA binding / CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / detection of maltose stimulus / maltose transport complex / carbohydrate transport / establishment of mitotic spindle orientation / carbohydrate transmembrane transporter activity / maltose binding / mitotic cytokinesis / negative regulation of tumor necrosis factor-mediated signaling pathway / maltose transport / maltodextrin transmembrane 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 / heterochromatin organization / Packaging Of Telomere Ends / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / 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 / nucleosomal DNA binding / 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 / Assembly of the ORC complex at the origin of replication / ATP-binding cassette (ABC) transporter complex / SUMOylation of chromatin organization proteins / DNA methylation / cell chemotaxis / Condensation of Prophase Chromosomes / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / PRC2 methylates histones and DNA / innate immune response in mucosa / Defective pyroptosis / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / 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 / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / B-WICH complex positively regulates rRNA expression / G2/M DNA damage checkpoint / HDMs demethylate histones / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / PKMTs methylate histone lysines / Meiotic recombination / kinetochore / RMTs methylate histone arginines / Pre-NOTCH Transcription and Translation / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / antimicrobial humoral immune response mediated by antimicrobial peptide / UCH proteinases / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / HATs acetylate histones / gene expression / Processing of DNA double-strand break ends / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / outer membrane-bounded periplasmic space / Oxidative Stress Induced Senescence / defense response to Gram-negative bacterium Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() ![]() unidentified cloning vector (others) synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å | ||||||||||||
![]() | Ariyoshi, M. / Makino, F. / Fukagawa, T. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 320.9 KB | Display | ![]() |
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PDB format | ![]() | 239.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 904.6 KB | Display | ![]() |
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Full document | ![]() | 925.6 KB | Display | |
Data in XML | ![]() | 39 KB | Display | |
Data in CIF | ![]() | 61.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 30239MC ![]() 7bxtC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 1.6 TB Data #1: CENP-A nucleosome in complex with phosphorylated CENP-C C-terminal domain (601-864) and CENP-N N-terminal domain (1-211) [micrographs - multiframe]) |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 5 types, 10 molecules AEBFCGDHKL
#1: Protein | Mass: 16089.938 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: chimeric protein of chicken Histone H3.2 (RESIDUES 1-64) and chicken CENP-C (RESIDUES 65-141, UNP RESIDUES 55-131) Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 11337.374 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Protein | Mass: 14165.551 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #4: Protein | Mass: 13935.239 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #7: Protein | Mass: 73796.312 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: fusion protein of N-terminal MBP (RESIDUES 201-600), linkers, and the phosphorylated C-terminal chicken CNPE-C (RESIDUES 612-864, UNP RESIDUES 601-853) Source: (gene. exp.) unidentified cloning vector (others), (gene. exp.) ![]() ![]() Variant: cloning vector pMal-C5X / Plasmid: pMAL_c2X / Production host: ![]() ![]() |
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-DNA chain , 2 types, 2 molecules IJ
#5: DNA chain | Mass: 44520.383 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#6: DNA chain | Mass: 44991.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 0.33 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||||||||||||||
Source (natural) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: MOLYBDENUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R0.6/1 | ||||||||||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
Microscopy | Model: JEOL CRYO ARM 200 |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER / Temperature (max): 80 K / Temperature (min): 80 K |
Image recording | Average 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 scans | Sampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50 |
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Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 343497 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38542 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE |