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- EMDB-33666: chicken KNL2 in complex with the CENP-A nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-33666
Titlechicken KNL2 in complex with the CENP-A nucleosome
Map data
Sample
  • Complex: the CENP-C like motif of chicken KNL2 in complex with the CENP-A nucleosome
    • Complex: chimeric CENP-A
      • Protein or peptide: Histone H3.1,Histone H3-like centromeric protein A
    • Complex: nucleosome
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 1-B/E
      • Protein or peptide: Histone H2B type 1-J
    • Complex: DNA
      • DNA: Chains: I
      • DNA: Chains: J
    • Complex: Myb-like domain-containing protein
      • Protein or peptide: Myb-like domain-containing protein
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsAriyoshi M / Jiang H / Makino F / Fukagawa T
Funding support Japan, 7 items
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, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33666.png

Downloads & links

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Map

FileDownload / File: emd_33666.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.98 Å
Density
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.13443454 - 0.19643733
Average (Standard dev.)0.00057929126 (±0.009506193)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 188.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33666_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33666_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : the CENP-C like motif of chicken KNL2 in complex with the CENP-A ...

EntireName: the CENP-C like motif of chicken KNL2 in complex with the CENP-A nucleosome
Components
  • Complex: the CENP-C like motif of chicken KNL2 in complex with the CENP-A nucleosome
    • Complex: chimeric CENP-A
      • Protein or peptide: Histone H3.1,Histone H3-like centromeric protein A
    • Complex: nucleosome
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 1-B/E
      • Protein or peptide: Histone H2B type 1-J
    • Complex: DNA
      • DNA: Chains: I
      • DNA: Chains: J
    • Complex: Myb-like domain-containing protein
      • Protein or peptide: Myb-like domain-containing protein

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Supramolecule #1: the CENP-C like motif of chicken KNL2 in complex with the CENP-A ...

SupramoleculeName: the CENP-C like motif of chicken KNL2 in complex with the CENP-A nucleosome
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: The 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.
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 200 KDa

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Supramolecule #2: chimeric CENP-A

SupramoleculeName: chimeric CENP-A / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: nucleosome

SupramoleculeName: nucleosome / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #5-#6

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Supramolecule #5: Myb-like domain-containing protein

SupramoleculeName: Myb-like domain-containing protein / type: complex / Chimera: Yes / ID: 5 / Parent: 1 / Macromolecule list: #7

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Macromolecule #1: Histone H3.1,Histone H3-like centromeric protein A

MacromoleculeName: Histone H3.1,Histone H3-like centromeric protein A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 16.372217 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMARTKQT ARKSTGGKAP RKQLATKAAR KSAPATGGVK KPHRYRPGTV ALREIRRYQK STELLIRRQP FARVVREICL LFTRGVDYR WQAMALLALQ EAAEAFLVHL LEDAYLCSLH ARRVTLYPKD LQLARRLRGL QGEGF

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.676703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMSGRGKG GKGLGKGGAK RHRKVLRDNI QGITKPAIRR LARRGGVKRI SGLIYEETRG VLKVFLENVI RDAVTYTEHA KRKTVTAMD VVYALKRQGR TLYGFGG

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Macromolecule #3: Histone H2A type 1-B/E

MacromoleculeName: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.165551 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK ARAKAKTRSS RAGLQFPVGR VHRLLRKGNY SERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIR NDEELNKLLG RVTIAQGGVL PNIQAVLLPK KTESHHKAKG K

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Macromolecule #4: Histone H2B type 1-J

MacromoleculeName: Histone H2B type 1-J / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.217516 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMPEPAKS APAPKKGSKK AVTKAQKKDG KKRKRSRKES YSIYVYKVLK QVHPDTGISS KAMGIMNSFV NDIFERIAGE ASRLAHYNK RSTITSREIQ TAVRLLLPGE LAKHAVSEGT KAVTKYTSAK

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Macromolecule #7: Myb-like domain-containing protein

MacromoleculeName: Myb-like domain-containing protein / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 4.983576 KDa
SequenceString:
SSNGIYTRSG RLVKPPLSFW CGEREFVDRE LNVTIQKGGT DYLS

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Macromolecule #5: Chains: I

MacromoleculeName: Chains: I / type: dna / ID: 5
Details: extracted from Escherichia coli as synthetic construct (plasmid) and purified
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 43.911992 KDa
SequenceString: (DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC)(DC) (DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG)(DG) (DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA)(DT) (DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG)(DA) (DC) (DA)(DG)(DC)(DT)(DC)(DT) ...String:
(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC)(DC) (DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG)(DG) (DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA)(DT) (DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG)(DA) (DC) (DA)(DG)(DC)(DT)(DC)(DT)(DA)(DG) (DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA)(DA) (DA)(DC) (DG)(DC)(DA)(DC)(DG)(DT)(DA) (DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC)(DC) (DC)(DC)(DC) (DG)(DC)(DG)(DT)(DT)(DT) (DT)(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DA)(DA) (DG)(DG)(DG)(DG) (DA)(DT)(DT)(DA)(DC) (DT)(DC)(DC)(DC)(DT)(DA)(DG)(DT)(DC)(DT) (DC)(DC)(DA)(DG)(DG) (DC)(DA)(DC)(DG) (DA)(DG)(DT)(DC)(DA)(DG)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DC)(DA)(DT) (DC)(DG)(DA)

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Macromolecule #6: Chains: J

MacromoleculeName: Chains: J / type: dna / ID: 6
Details: extracted from Escherichia coli as synthetic construct (plasmid) and purified
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.365242 KDa
SequenceString: (DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DT)(DC) (DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG)(DA) (DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA)(DG) (DT) (DA)(DA)(DT)(DC)(DC)(DC) ...String:
(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DT)(DC) (DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG)(DA) (DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA)(DG) (DT) (DA)(DA)(DT)(DC)(DC)(DC)(DC)(DT) (DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT)(DA)(DA) (DA)(DA) (DC)(DG)(DC)(DG)(DG)(DG)(DG) (DG)(DA)(DC)(DA)(DG)(DC)(DG)(DC)(DG)(DT) (DA)(DC)(DG) (DT)(DG)(DC)(DG)(DT)(DT) (DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG)(DC) (DT)(DA)(DG)(DA) (DG)(DC)(DT)(DG)(DT) (DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA)(DA) (DT)(DT)(DG)(DA)(DG) (DC)(DG)(DG)(DC) (DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC)(DG) (DG)(DG)(DA)(DT)(DT)(DC) (DT)(DG)(DA)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
0.1 %C32H58N2O7SCHAPS
5.0 mMC9H15O6PTCEP
20.0 mMC8H18N2O4SHEPES
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Calibrated magnification: 49000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5858 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number real images: 9450 / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber selected: 10331795
CTF correctionSoftware - Name: Gctf (ver. 9.03)
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 300000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 270464
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5z23


7by0

chain_id: K, residue_range: 649-679
DetailsInitial local fitting and refinement were performed using PHENIX. The complex model was iteratively modified in Coot and refined in PHENIX.
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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