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- EMDB-20839: Structure of a Yeast Centromeric Nucleosome at 2.7 Angstrom resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-20839
TitleStructure of a Yeast Centromeric Nucleosome at 2.7 Angstrom resolution
Map dataSharpened and masked map of CEN-NCP contoured at 0.16 level
Sample
  • Complex: Yeast Centromeric Nucleosome
    • Complex: Histone H3-like centromeric protein CSE4
      • Protein or peptide: Histone H3-like centromeric protein CSE4
    • Complex: Histone H4, Histone H2A, Histone H2B.1
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B.1
    • Complex: DNA
      • DNA: DNA (119-MER)
      • DNA: DNA (119-MER)
Function / homology
Function and homology information


2-micrometer circle DNA / 2-micrometer plasmid partitioning / centromeric DNA binding / kinetochore assembly / condensed chromosome, centromeric region / mitotic sister chromatid segregation / rRNA transcription / protein localization to CENP-A containing chromatin / CENP-A containing nucleosome / structural constituent of chromatin ...2-micrometer circle DNA / 2-micrometer plasmid partitioning / centromeric DNA binding / kinetochore assembly / condensed chromosome, centromeric region / mitotic sister chromatid segregation / rRNA transcription / protein localization to CENP-A containing chromatin / CENP-A containing nucleosome / structural constituent of chromatin / nucleosome / sequence-specific DNA binding / protein heterodimerization activity / DNA repair / DNA binding / nucleus
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 H4, conserved site ...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 / 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 H3-like centromeric protein CSE4 / Histone H2A / Histone H2B.1 / Histone H4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsMigl D / Kschonsak M / Arthur CP / Khin Y / Harrison SC / Ciferri C / Dimitrova YN
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM62580 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Structure / Year: 2020
Title: Cryoelectron Microscopy Structure of a Yeast Centromeric Nucleosome at 2.7 Å Resolution.
Authors: David Migl / Marc Kschonsak / Christopher P Arthur / Yadana Khin / Stephen C Harrison / Claudio Ciferri / Yoana N Dimitrova /
Abstract: Kinetochores mediate chromosome segregation during cell division. They assemble on centromeric nucleosomes and capture spindle microtubules. In budding yeast, a kinetochore links a single nucleosome, ...Kinetochores mediate chromosome segregation during cell division. They assemble on centromeric nucleosomes and capture spindle microtubules. In budding yeast, a kinetochore links a single nucleosome, containing the histone variant Cse4 instead of H3, with a single microtubule. Conservation of most kinetochore components from yeast to metazoans suggests that the yeast kinetochore represents a module of the more complex metazoan arrangements. We describe here a streamlined protocol for reconstituting a yeast centromeric nucleosome and a systematic exploration of cryo-grid preparation. These developments allowed us to obtain a high-resolution cryoelectron microscopy reconstruction. As suggested by previous work, fewer base pairs are in tight association with the histone octamer than there are in canonical nucleosomes. Weak binding of the end DNA sequences may contribute to specific recognition by other inner kinetochore components. The centromeric nucleosome structure and the strategies we describe will facilitate studies of many other aspects of kinetochore assembly and chromatin biochemistry.
History
DepositionOct 17, 2019-
Header (metadata) releaseNov 6, 2019-
Map releaseNov 6, 2019-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uph
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20839.png

Downloads & links

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Map

FileDownload / File: emd_20839.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and masked map of CEN-NCP contoured at 0.16 level
Voxel sizeX=Y=Z: 0.849 Å
Density
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.31615224 - 0.71833694
Average (Standard dev.)0.032766916 (±0.036843054)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 163.008 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8490.8490.849
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z163.008163.008163.008
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.3160.7180.033

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Supplemental data

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Additional map: Gaussian filtered unmasked map

Fileemd_20839_additional_1.map
AnnotationGaussian filtered unmasked map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked map of CEN-NCP contoured at 1.1 level

Fileemd_20839_additional_2.map
AnnotationMasked map of CEN-NCP contoured at 1.1 level
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmasked map of CEN-NCP contoured at 1.3 level

Fileemd_20839_additional_3.map
AnnotationUnmasked map of CEN-NCP contoured at 1.3 level
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Masked halfmap 2

Fileemd_20839_half_map_1.map
AnnotationMasked halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Masked halfmap 1

Fileemd_20839_half_map_2.map
AnnotationMasked halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast Centromeric Nucleosome

EntireName: Yeast Centromeric Nucleosome
Components
  • Complex: Yeast Centromeric Nucleosome
    • Complex: Histone H3-like centromeric protein CSE4
      • Protein or peptide: Histone H3-like centromeric protein CSE4
    • Complex: Histone H4, Histone H2A, Histone H2B.1
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B.1
    • Complex: DNA
      • DNA: DNA (119-MER)
      • DNA: DNA (119-MER)

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Supramolecule #1: Yeast Centromeric Nucleosome

SupramoleculeName: Yeast Centromeric Nucleosome / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 240 kDa/nm

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Supramolecule #2: Histone H3-like centromeric protein CSE4

SupramoleculeName: Histone H3-like centromeric protein CSE4 / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Histone H4, Histone H2A, Histone H2B.1

SupramoleculeName: Histone H4, Histone H2A, Histone H2B.1 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Source (natural)Organism: Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)
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
Source (natural)Organism: unidentified (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Histone H3-like centromeric protein CSE4

MacromoleculeName: Histone H3-like centromeric protein CSE4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 26.885434 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSKQQWVSS AIQSDSSGRS LSNVNRLAGD QQSINDRALS LLQRTRATKN LFPRREERRR YESSKSDLDI ETDYEDQAGN LEIETENEE EAEMETEVPA PVRTHSYALD RYVRQKRREK QRKQSLKRVE KKYTPSELAL YEIRKYQRST DLLISKIPFA R LVKEVTDE ...String:
MSSKQQWVSS AIQSDSSGRS LSNVNRLAGD QQSINDRALS LLQRTRATKN LFPRREERRR YESSKSDLDI ETDYEDQAGN LEIETENEE EAEMETEVPA PVRTHSYALD RYVRQKRREK QRKQSLKRVE KKYTPSELAL YEIRKYQRST DLLISKIPFA R LVKEVTDE FTTKDQDLRW QSMAIMALQE ASEAYLVGLL EHTNLLALHA KRITIMKKDM QLARRIRGQF I

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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: Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)
Strain: ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37
Molecular weightTheoretical: 13.332434 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHENLY FQSNAMSGRG KGGKGLGKGG AKRHRKILRD NIQGITKPAI RRLARRGGVK RISGLIYEEV RNVLKTFLES VIRDAVTYT EHAKRKTVTS LDVVYALKRQ GRTLYGFGG

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Macromolecule #3: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)
Strain: ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37
Molecular weightTheoretical: 15.739005 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHENLY FQSNAMSGKG GKAGSAAKAS QSRSAKAGLT FPVGRVHRLL RKGNYAQRIG SGAPVYLTAV LEYLAAEILE LAGNAARDN KKTRIIPRHL QLAIRNDDEL NKLLGNVTIA QGGVLPNIHQ NLLPKKSSKA KASQEL

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Macromolecule #4: Histone H2B.1

MacromoleculeName: Histone H2B.1 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)
Strain: ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37
Molecular weightTheoretical: 16.219361 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHENLY FQSNAMSAKA SKAPASKAPA EKKPAAKKTS SSTDPSKKRT KARKETYSSY IYKVLKQTHP DTGISQKSMS ILNSFVNDI FERIATESSK LAAYNKKSTI SAREIQTAVR LILPGELAKH AVSEGTRAVT KYSSSTQA

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Macromolecule #5: DNA (119-MER)

MacromoleculeName: DNA (119-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 45.13877 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #6: DNA (119-MER)

MacromoleculeName: DNA (119-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 45.610043 KDa
SequenceString: (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) ...String:
(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)

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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 mg/mL
BufferpH: 7.5
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.249 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 265380

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