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- PDB-7y7i: chicken KNL2 in complex with the CENP-A nucleosome -

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Basic information

Entry
Database: PDB / ID: 7y7i
Titlechicken KNL2 in complex with the CENP-A nucleosome
Components
  • Chains: I
  • Chains: J
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1,Histone H3-like centromeric protein A
  • Histone H4
  • Myb-like domain-containing protein
KeywordsNUCLEAR PROTEIN/DNA / centromere / kinetochore / NUCLEAR PROTEIN-DNA COMPLEX
Function / homology
Function and homology information


CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes ...CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / epigenetic regulation of gene expression / 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 / Interleukin-7 signaling / SUMOylation of chromatin organization proteins / Assembly of the ORC complex at the origin of replication / 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 / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / 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 / 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 / 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 / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / Factors involved in megakaryocyte development and platelet production / Processing of DNA double-strand break ends / HATs acetylate histones / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / killing of cells of another organism / Estrogen-dependent gene expression / defense response to Gram-negative bacterium / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / cadherin binding / Amyloid fiber formation / protein heterodimerization activity / negative regulation of cell population proliferation / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / nucleus / cytosol
Similarity search - Function
SANT associated / KNL2-like / SANTA (SANT Associated) / Myb-like domain profile. / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...SANT associated / KNL2-like / SANTA (SANT Associated) / Myb-like domain profile. / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / 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 / 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 / Homeobox-like domain superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Myb-like domain-containing protein / Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1 / Histone H3-like centromeric protein A
Similarity search - Component
Biological speciesGallus gallus (chicken)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsAriyoshi, M. / Jiang, H. / Makino, F. / Fukagawa, T.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22H00408 Japan
Japan Society for the Promotion of Science (JSPS)21K06048 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101117 Japan
Japan Agency for Medical Research and Development (AMED)JP17pc0101020 Japan
Japan Science and TechnologyJPMJCR21E6 Japan
Japan Society for the Promotion of Science (JSPS)22H04692 Japan
Japan Society for the Promotion of Science (JSPS)21H05752 Japan
CitationJournal: Biorxiv / Year: 2022
Title: The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2.
Authors: Jiang, H. / Ariyoshi, M. / Watanabe, R. / Makino, F. / Namba, K. / Fukagawa, T.
History
DepositionJun 22, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.1,Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.1,Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: Chains: I
J: Chains: J
K: Myb-like domain-containing protein
L: Myb-like domain-containing protein


Theoretical massNumber of molelcules
Total (without water)211,10812
Polymers211,10812
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3.1,Histone H3-like centromeric protein A / Histone H3/l / CENP-A


Mass: 16372.217 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: H3C1, CENPA / Production host: Escherichia coli (E. coli) / References: UniProt: P68431, UniProt: Q6XXM1
#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) / Gene: H4C1 / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E


Mass: 14165.551 Da / Num. of mol.: 2 / Mutation: L51M, L58M, L93M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / 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: 14217.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BJ / Production host: Escherichia coli (E. coli) / References: UniProt: P06899

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain Chains: I


Mass: 43911.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: extracted from Escherichia coli as synthetic construct (plasmid) and purified
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain Chains: J


Mass: 44365.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: extracted from Escherichia coli as synthetic construct (plasmid) and purified
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Protein/peptide , 1 types, 2 molecules KL

#7: Protein/peptide Myb-like domain-containing protein


Mass: 4983.576 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Gallus gallus (chicken) / References: UniProt: F1NE16

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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
IDNameTypeDetailsEntity IDParent-IDSource
1the CENP-C like motif of chicken KNL2 in complex with the CENP-A nucleosomeCOMPLEXThe CENP-C like motif peptide of chicken KNL2 was prepared by peptide synthesis. The CENP-A nucleosome was reconstituted in vitro using recombinant proteins, incubated with the KNL2 peptide and concentrated.all0MULTIPLE SOURCES
2chimeric CENP-ACOMPLEX#11RECOMBINANT
3nucleosomeCOMPLEX#2-#41RECOMBINANT
4DNACOMPLEX#5-#61RECOMBINANT
5Myb-like domain-containing proteinCOMPLEX#71SYNTHETIC
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Gallus gallus (chicken)9031
31Gallus gallus (chicken)9031
42Homo sapiens (human)9606
53synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
31Escherichia coli (E. coli)562
42Escherichia coli (E. coli)562
53Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
20.1 %CHAPSC32H58N2O7S1
35 mMTCEPC9H15O6P1
420 mMHEPESC8H18N2O4S1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 49000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 9450
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5858 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.9image acquisition
4Gctf9.03CTF correction
7PHENIX1.20.1model fittingrigid body refinement
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.20.1model refinementreal space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10331795
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270464 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Initial local fitting and refinement were performed using PHENIX. The complex model was iteratively modified in Coot and refined in PHENIX.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15Z23

5z23

1
27BY0

7by0

K1649-679
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 66.92 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003513258
ELECTRON MICROSCOPYf_angle_d0.565519135
ELECTRON MICROSCOPYf_chiral_restr0.03552165
ELECTRON MICROSCOPYf_plane_restr0.00371429
ELECTRON MICROSCOPYf_dihedral_angle_d31.8173804

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