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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
TitleThe cryo-EM structure of CENP-A nucleosome in complex with the phosphorylated CENP-C
Components
  • (DNA (145-MER)) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.2,Histone H3-like centromeric protein A
  • Histone H4
  • Maltose Binding Protein tag, linker,CENP-C
KeywordsCELL CYCLE / CENP-A nucleosome / CENP-C / CENP-N / complex / kinetochore / NUCLEAR PROTEIN
Function / homology
Function and homology information


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 / 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 ...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 / 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 / Regulation of endogenous retroelements by KRAB-ZFP proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / B-WICH complex positively regulates rRNA 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 / establishment of mitotic spindle orientation / maltose binding / carbohydrate transport / maltose transport / maltodextrin transmembrane transport / mitotic cytokinesis / carbohydrate transmembrane transporter activity / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / 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 / 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 / telomere organization / Meiotic synapsis / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / ATP-binding cassette (ABC) transporter complex / DNA methylation / cell chemotaxis / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / innate immune response in mucosa / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / 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 / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / heterochromatin formation / PKMTs methylate histone lysines / Metalloprotease DUBs / Meiotic recombination / kinetochore / Pre-NOTCH Transcription and Translation / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / antimicrobial humoral immune response mediated by antimicrobial peptide / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / antibacterial humoral response / 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 / Processing of DNA double-strand break ends
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 / 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 ...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 / 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 / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / RmlC-like jelly roll fold / Histone H2A / Histone 2A / 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) / CENP-C / Histone H2A type 1-B/E / Histone H2B type 1-J / Maltose/maltodextrin-binding periplasmic protein / Histone H4 / Histone H3.2 / 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: 4.5 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 21, 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 1.2Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2,Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.2,Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (145-MER)
J: DNA (145-MER)
K: Maltose Binding Protein tag, linker,CENP-C
L: Maltose Binding Protein tag, linker,CENP-C


Theoretical massNumber of molelcules
Total (without water)348,16112
Polymers348,16112
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area59060 Å2
ΔGint-393 kcal/mol
Surface area78970 Å2

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Components

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Protein , 5 types, 10 molecules AEBFCGDHKL

#1: Protein Histone H3.2,Histone H3-like centromeric protein A / Centromere protein A / GgCENP-A


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.) Gallus gallus (chicken) / Plasmid: pET15b / Gene: CENPA / Production host: Escherichia coli (E. coli) / References: UniProt: Q6XXM1, UniProt: P84229*PLUS
#2: Protein Histone H4


Mass: 11337.374 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: 14165.551 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: P04908
#4: Protein Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 13935.239 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BJ, H2BFR / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / References: UniProt: P06899
#7: Protein Maltose Binding Protein tag, linker,CENP-C


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.) Gallus gallus (chicken)
Variant: cloning vector pMal-C5X / Plasmid: pMAL_c2X / Production host: Escherichia coli (E. coli) / References: UniProt: O57393, UniProt: P0AEX9*PLUS

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-MER)


Mass: 44520.383 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (145-MER)


Mass: 44991.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Details

Has ligand of interestY
Has protein modificationY

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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 C-terminal 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 1-JCOMPLEX#41MULTIPLE SOURCES
6DNA (145-mer)COMPLEX#5-#61MULTIPLE SOURCES
7Maltodextrin-binding protein,LINKER PEPTIDE,CENP-CCOMPLEX#71MULTIPLE SOURCES
Molecular weightValue: 0.33 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Gallus gallus (chicken)9031
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
75Gallus gallus (chicken)9031
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaCl1
32 mMDTTC4H10O2S21
41 mMEDTAC10H16N2O81
50.1 %CHAPSC32H58N2O7S1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
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: 343497
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38542 / Symmetry type: POINT
RefinementCross valid method: NONE

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