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- EMDB-0586: Cryo-EM structure of the centromeric nucleosome with native alpha... -

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Entry
Database: EMDB / ID: EMD-0586
TitleCryo-EM structure of the centromeric nucleosome with native alpha satellite DNA
Map data
SampleCENP-A nucleosome with native alpha satellite DNA sequence:
Histone H3-like centromeric protein A / Histone H4 / Histone H2A type 1-B/E / Histone H2B type 1-J / (nucleic-acidNucleic acid) x 2
Function / homology
Function and homology information


Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Senescence-Associated Secretory Phenotype (SASP) / 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 / Pre-NOTCH Transcription and Translation / Packaging Of Telomere Ends ...Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Senescence-Associated Secretory Phenotype (SASP) / 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 / Pre-NOTCH Transcription and Translation / Packaging Of Telomere Ends / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Meiotic synapsis / Cleavage of the damaged purine / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged pyrimidine / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / PKMTs methylate histone lysines / HDMs demethylate histones / 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 Promoter Escape / 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) / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Metalloprotease DUBs / Ub-specific processing proteases / SUMOylation of chromatin organization proteins / 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 / 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 / UCH proteinases / condensed chromosome inner kinetochore / kinetochore assembly / protein localization to chromosome, centromeric region / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / establishment of mitotic spindle orientation / negative regulation of megakaryocyte differentiation / condensed nuclear chromosome, centromeric region / CENP-A containing nucleosome assembly / chromosome, centromeric region / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / mitotic cytokinesis / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / nuclear nucleosome / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / nucleosomal DNA binding / DNA-templated transcription, initiation / regulation of gene silencing by miRNA / nuclear chromosome / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / lipopolysaccharide binding / protein heterotetramerization / chromatin organization / double-strand break repair via nonhomologous end joining / antibacterial humoral response / nuclear chromosome, telomeric region / killing of cells of other organism / antimicrobial humoral immune response mediated by antimicrobial peptide / nuclear chromatin / protein ubiquitination / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / protein domain specific binding / chromatin binding / viral process / cellular protein metabolic process / protein heterodimerization activity / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / nucleoplasm
Histone H4, conserved site / Histone H3/CENP-A / Histone H2B signature. / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone-fold / Histone H3 signature 2. ...Histone H4, conserved site / Histone H3/CENP-A / Histone H2B signature. / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone-fold / Histone H3 signature 2. / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / Centromere kinetochore component CENP-T histone fold / C-terminus of histone H2A / Histone H2A signature. / Histone H4 signature.
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H3-like centromeric protein A / Histone H4
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.395 Å
AuthorsZhou B-R
CitationJournal: Nat Commun / Year: 2019
Title: Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment.
Authors: Bing-Rui Zhou / K N Sathish Yadav / Mario Borgnia / Jingjun Hong / Baohua Cao / Ada L Olins / Donald E Olins / Yawen Bai / Ping Zhang /
Abstract: Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a ...Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.
Validation ReportPDB-ID: 6o1d

SummaryFull reportAbout validation report
DateDeposition: Feb 19, 2019 / Header (metadata) release: Oct 10, 2018 / Map release: May 22, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6o1d
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0586.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.28 Å
0.85 Å/pix.
x 256 pix.
= 217.28 Å
0.85 Å/pix.
x 256 pix.
= 217.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84875 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.054393053 - 0.08594776
Average (Standard dev.)-0.000007443697 (±0.0027541937)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.848750.848750.84875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z217.280217.280217.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0540.086-0.000

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Supplemental data

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Sample components

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Entire CENP-A nucleosome with native alpha satellite DNA sequence

EntireName: CENP-A nucleosome with native alpha satellite DNA sequence
Number of components: 7

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Component #1: protein, CENP-A nucleosome with native alpha satellite DNA sequence

ProteinName: CENP-A nucleosome with native alpha satellite DNA sequence
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 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 18.038818 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.394426 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Histone H2A type 1-B/E

ProteinName: Histone H2A type 1-B/E / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.165551 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Histone H2B type 1-J

ProteinName: Histone H2B type 1-J / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.935239 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: nucleic-acid, DNA (145-MER)

nucleic acidName: DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC)(DC) (DA)(DT)(DC)(DA)(DA)(DA)(DA)(DG)(DG)(DC) (DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DC)(DT)(DG)(DT)(DG)(DA)(DG)(DT)(DG)(DA) (DA)(DA)(DC)(DT)(DC)(DC)(DA)(DT)(DC)(DA) (DT)(DC)(DA)(DC)(DA)(DA)(DA)(DG)(DA)(DA) (DT)(DA)(DT)(DT)(DC)(DT)(DG)(DA)(DG)(DA) (DA)(DT)(DG)(DC)(DT)(DT)(DC)(DC)(DG)(DT) (DT)(DT)(DG)(DC)(DC)(DT)(DT)(DT)(DT)(DA) (DT)(DA)(DT)(DG)(DA)(DA)(DC)(DT)(DT)(DC) (DC)(DT)(DG)(DA)(DT)
MassTheoretical: 44.539535 kDa
SourceSpecies: Homo sapiens (human)

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Component #7: nucleic-acid, DNA (145-MER)

nucleic acidName: DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DA)(DG)(DG)(DA)(DA)(DG)(DT) (DT)(DC)(DA)(DT)(DA)(DT)(DA)(DA)(DA)(DA) (DG)(DG)(DC)(DA)(DA)(DA)(DC)(DG)(DG)(DA) (DA)(DG)(DC)(DA)(DT)(DT)(DC)(DT)(DC)(DA) (DG)(DA)(DA)(DT)(DA)(DT)(DT)(DC)(DT)(DT) (DT)(DG)(DT)(DG)(DA)(DT)(DG)(DA)(DT)(DG) (DG)(DA)(DG)(DT)(DT)(DT)(DC)(DA)(DC)(DT) (DC)(DA)(DC)(DA)(DG)(DA)(DG)(DC)(DT)(DG) (DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT)(DT) (DT)(DT)(DG)(DA)(DT)(DG)(DG)(DA)(DG)(DC) (DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA)(DA) (DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DA) (DT)(DT)(DG)(DA)(DT)
MassTheoretical: 44.948793 kDa
SourceSpecies: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.5 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Details: blot for 2.5 sec before plunging.

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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 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1256
Details: Images were collected in movie-mode at 38 frames over 15.2 seconds

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 303863
3D reconstructionSoftware: RELION / Resolution: 3.395 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 6E0P
Output model

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