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

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Basic information

Entry
Database: EMDB / ID: EMD-0586
TitleCryo-EM structure of the centromeric nucleosome with native alpha satellite DNA
Map dataMain EM map after sharpening
Sample
  • Complex: CENP-A nucleosome with native alpha satellite DNA sequence
    • Protein or peptide: Histone H3-like centromeric protein A
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
KeywordsCENP-A / Centromere / Native alpha satellite DNA / nucleosome / NUCLEAR PROTEIN
Function / homology
Function and homology information


CENP-A containing chromatin assembly / kinetochore assembly / protein localization to chromosome, centromeric region / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin ...CENP-A containing chromatin assembly / kinetochore assembly / protein localization to chromosome, centromeric region / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / pericentric heterochromatin / 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 / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Deposition of new CENPA-containing nucleosomes at the centromere / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / telomere organization / Inhibition of DNA recombination at telomere / Meiotic synapsis / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / Resolution of Sister Chromatid Cohesion / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / innate immune response in mucosa / SUMOylation of chromatin organization proteins / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / 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 / RHO GTPases Activate Formins / 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 / G2/M DNA damage checkpoint / HDMs demethylate histones / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / HCMV Early Events / antimicrobial humoral immune response mediated by antimicrobial peptide / structural constituent of chromatin / Separation of Sister Chromatids / antibacterial humoral response / UCH proteinases / nucleosome / heterochromatin formation / E3 ubiquitin ligases ubiquitinate target proteins / nucleosome assembly / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / defense response to Gram-negative bacterium / Oxidative Stress Induced Senescence / killing of cells of another organism / Estrogen-dependent gene expression / chromosome, telomeric region / defense response to Gram-positive bacterium / Ub-specific processing proteases / Amyloid fiber formation / protein heterodimerization activity / negative regulation of cell population proliferation / chromatin binding / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / nucleus / membrane / cytosol
Similarity search - Function
: / 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 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 / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / 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
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H3-like centromeric protein A / Histone H4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.395 Å
AuthorsZhou B-R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)the intramural research program United States
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.
History
Header (metadata) releaseOct 10, 2018-
DepositionFeb 19, 2019-
Map releaseMay 22, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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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

emd_0586.png

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)
AnnotationMain EM map after sharpening
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

Histogram

Histogram (log scale)
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
Components
  • Complex: CENP-A nucleosome with native alpha satellite DNA sequence
    • Protein or peptide: Histone H3-like centromeric protein A
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)

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Supramolecule #1: CENP-A nucleosome with native alpha satellite DNA sequence

SupramoleculeName: CENP-A nucleosome with native alpha satellite DNA sequence
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Histone H3-like centromeric protein A

MacromoleculeName: Histone H3-like centromeric protein A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.038818 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSHHHHHH SSGLPRSHMG PRRRSRKPEA PRRRSPSPTP TPGPSRRGPS LGASSHQHSR RRQGWLKEIR KLQKSTHLLI RKLPFSRLA REICVKFTRG VDFNWQAQAL LALQEAAEAF LVHLFEDAYL LTLHAGRVTL FPKDVQLARR IRGLEEGLG

UniProtKB: Histone H3-like centromeric protein A

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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: 11.394426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVY ALKRQGRTLY GFGG

UniProtKB: Histone H4

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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

UniProtKB: Histone H2A type 1-B/E

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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: 13.935239 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSI YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSAK

UniProtKB: Histone H2B type 1-J

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Macromolecule #5: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.539535 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #6: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.948793 KDa
SequenceString: (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) ...String:
(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)

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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

Concentration1.5 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: blot for 2.5 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 2-38 / Number grids imaged: 1 / Number real images: 1256 / Average exposure time: 15.2 sec. / Average electron dose: 40.0 e/Å2
Details: Images were collected in movie-mode at 38 frames over 15.2 seconds
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.395 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0 beta2) / Number images used: 303863
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta2)
Final 3D classificationNumber classes: 4 / Avg.num./class: 75000 / Software - Name: RELION (ver. 3.0 beta2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6e0p


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6o1d:
Cryo-EM structure of the centromeric nucleosome with native alpha satellite DNA

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