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- EMDB-7293: Cryo-EM structure of CENP-A nucleosome in complex with kinetochor... -

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Entry
Database: EMDB / ID: 7293
TitleCryo-EM structure of CENP-A nucleosome in complex with kinetochore protein CENP-N
Map dataCryo-EM density map of CENP-A nucleosome in complex with kinetochore protein CENP-N
SampleCentromeric nucleosome in complex with kinetochore protein CENP-N
  • Histone H3-like centromeric protein A
  • Histone H4
  • Histone H2A
  • Histone H2B
  • (nucleic-acidNucleic acid) x 2
  • Maltose-binding periplasmic protein, Centromere protein N chimera
Function / homologySolute-binding family 1, conserved site / C-terminus of histone H2A / Oxidative Stress Induced Senescence / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / Formation of the beta-catenin:TCF transactivating complex / Packaging Of Telomere Ends / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal ...Solute-binding family 1, conserved site / C-terminus of histone H2A / Oxidative Stress Induced Senescence / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / Formation of the beta-catenin:TCF transactivating complex / Packaging Of Telomere Ends / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Meiotic synapsis / Bacterial extracellular solute-binding proteins, family 1 signature. / Histone H3 signature 2. / Histone H2B signature. / Histone H4 signature. / Histone H2A signature. / Centromere kinetochore component CENP-T histone fold / Histone H2A conserved site / Bacterial extracellular solute-binding protein / Histone H4, conserved site / Histone-fold / Centromere protein Chl4/mis15/CENP-N / Histone H2A/H2B/H3 / CENP-T/Histone H4, histone fold / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / TATA box binding protein associated factor (TAF) / Bacterial extracellular solute-binding protein / Histone H2A / Histone H4 / Histone H2B / Histone H3/CENP-A / Core histone H2A/H2B/H3/H4 / Kinetochore protein CHL4 like / Senescence-Associated Secretory Phenotype (SASP) / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / Metalloprotease DUBs / Amyloid fiber formation / Meiotic recombination / Estrogen-dependent gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / RNA Polymerase I Chain Elongation / RNA Polymerase I Promoter Opening / G2/M DNA damage checkpoint / Mitotic Prometaphase / Deposition of new CENPA-containing nucleosomes at the centromere / Processing of DNA double-strand break ends / Nonhomologous End-Joining (NHEJ) / PKMTs methylate histone lysines / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Ub-specific processing proteases / UCH proteinases / HDMs demethylate histones / HATs acetylate histones / RMTs methylate histone arginines / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / NoRC negatively regulates rRNA expression / SUMOylation of chromatin organization proteins / B-WICH complex positively regulates rRNA expression / DNA methylation / Transcriptional regulation by small RNAs / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RHO GTPases Activate Formins / Histone H2A, C-terminal domain / condensed chromosome inner kinetochore / kinetochore assembly / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic spindle orientation / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / condensed nuclear chromosome, centromeric region / chromatin silencing / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / condensed chromosome kinetochore / carbohydrate transmembrane transporter activity / chromosome, centromeric region / innate immune response in mucosa / mitotic cytokinesis / telomere organization / DNA replication-dependent nucleosome assembly / nuclear nucleosome / nucleosomal DNA binding / chromatin silencing at rDNA / regulation of gene silencing by miRNA / negative regulation of gene expression, epigenetic / DNA-templated transcription, initiation / nuclear chromosome / chromosome segregation / regulation of megakaryocyte differentiation / nucleosome / double-strand break repair via nonhomologous end joining / lipopolysaccharide binding
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 3.92 Å resolution
AuthorsChittori S / Hong J
CitationJournal: Science / Year: 2018
Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N.
Authors: Sagar Chittori / Jingjun Hong / Hayden Saunders / Hanqiao Feng / Rodolfo Ghirlando / Alexander E Kelly / Yawen Bai / Sriram Subramaniam
Abstract: Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by ...Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. We report cryo-electron microscopy (cryo-EM), biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We show that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrate analogous interactions in , which are further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with the coevolution of CENP-N and CENP-A and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.
Validation ReportPDB-ID: 6buz

SummaryFull reportAbout validation report
DateDeposition: Dec 11, 2017 / Header (metadata) release: Dec 20, 2017 / Map release: Dec 20, 2017 / Last update: Jan 31, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6buz
  • Surface level: 0.0014
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7293.map.gz (map file in CCP4 format, 702465 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
560 pix
0.42 Å/pix.
= 235.2 Å
560 pix
0.42 Å/pix.
= 235.2 Å
560 pix
0.42 Å/pix.
= 235.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.42 Å
Density
Contour Level:0.0014 (by author), 0.0014 (movie #1):
Minimum - Maximum-0.0023961891 - 0.0057761674
Average (Standard dev.)4.2810665E-5 (0.00027144956)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions560560560
Origin000
Limit559559559
Spacing560560560
CellA=B=C: 235.2 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.420.420.42
M x/y/z560560560
origin x/y/z0.0000.0000.000
length x/y/z235.200235.200235.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS560560560
D min/max/mean-0.0020.0060.000

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Supplemental data

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Sample components

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Entire Centromeric nucleosome in complex with kinetochore protein CENP-N

EntireName: Centromeric nucleosome in complex with kinetochore protein CENP-N
Number of components: 8

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Component #1: protein, Centromeric nucleosome in complex with kinetochore prote...

ProteinName: Centromeric nucleosome in complex with kinetochore protein CENP-N
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Histone H3-like centromeric protein A

ProteinName: Histone H3-like centromeric protein A / Recombinant expression: No
MassTheoretical: 18.195002 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, Histone H4

ProteinName: Histone H4 / Recombinant expression: No
MassTheoretical: 11.394426 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Component #4: protein, Histone H2A

ProteinName: Histone H2A / Recombinant expression: No
MassTheoretical: 14.165551 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Histone H2B

ProteinName: Histone H2B / Recombinant expression: No
MassTheoretical: 13.935239 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: nucleic-acid, DNA (147-MER)

Nucleic-acidName: DNA (147-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DG)(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)(DT)(DG)(DT)(DC) (DA)(DG)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DC) (DA)(DT)(DC)(DC)(DG)(DA)(DT)
MassTheoretical: 45.13877 kDa

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Component #7: nucleic-acid, DNA (147-MER)

Nucleic-acidName: DNA (147-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC) (DA)(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)(DC)(DG)(DA)(DT)
MassTheoretical: 45.610043 kDa

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Component #8: protein, Maltose-binding periplasmic protein, Centromere protein ...

ProteinName: Maltose-binding periplasmic protein, Centromere protein N chimera
Recombinant expression: No
MassTheoretical: 75.607797 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.7 mg/ml / pH: 7.4
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 61749
3D reconstructionSoftware: RELION / Resolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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