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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


kinetochore => GO:0000776 / Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / RHO GTPases Activate Formins / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / HDACs deacetylate histones / HATs acetylate histones ...kinetochore => GO:0000776 / Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / RHO GTPases Activate Formins / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / HDACs deacetylate histones / HATs acetylate histones / 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 / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Regulation of endogenous retroelements by KRAB-ZFP proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / Resolution of Sister Chromatid Cohesion / B-WICH complex positively regulates rRNA expression / EML4 and NUDC in mitotic spindle formation / Deposition of new CENPA-containing nucleosomes at the centromere / Separation of Sister Chromatids / Factors involved in megakaryocyte development and platelet production / spindle attachment to meiosis I kinetochore / CENP-A containing chromatin assembly / centromeric DNA binding / kinetochore assembly / protein localization to chromosome, centromeric region / attachment of mitotic spindle microtubules to kinetochore / condensed chromosome, centromeric region / detection of maltose stimulus / maltose transport complex / carbohydrate transport / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / carbohydrate transmembrane transporter activity / maltose binding / 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 / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Inhibition of DNA recombination at telomere / Meiotic synapsis / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / ATP-binding cassette (ABC) transporter complex / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / innate immune response in mucosa / SUMOylation of chromatin organization proteins / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / cell chemotaxis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / chromosome segregation / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / 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 / G2/M DNA damage checkpoint / HDMs demethylate histones / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / kinetochore / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / HCMV Early Events / antimicrobial humoral immune response mediated by antimicrobial peptide / structural constituent of chromatin / antibacterial humoral response / UCH proteinases
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 / RmlC-like cupin domain superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein ...: / 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 / RmlC-like cupin domain superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / : / Histone H2B signature. / Histone H2B / Histone H2B / RmlC-like jelly roll fold / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / 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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Structure viewerMolecule:
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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
Water00
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 chlorideNaCl1
32 mMDTTC4H10O2S21
41 mMEDTAC10H16N2O81
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 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 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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