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- PDB-6uph: Structure of a Yeast Centromeric Nucleosome at 2.7 Angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 6uph
TitleStructure of a Yeast Centromeric Nucleosome at 2.7 Angstrom resolution
Components
  • (DNA (119-MER)) x 2
  • Histone H2A
  • Histone H2B.1
  • Histone H3-like centromeric protein CSE4
  • Histone H4
KeywordsCELL CYCLE / Histones / Nucleosome / Centromere / Kinetochore / Yeast
Function / homology
Function and homology information


protein localization to CENP-A containing chromatin / 2-micrometer circle DNA / 2-micrometer plasmid partitioning / CENP-A containing nucleosome / centromeric DNA binding / kinetochore assembly / condensed chromosome, centromeric region / kinetochore => GO:0000776 / mitotic sister chromatid segregation / kinetochore ...protein localization to CENP-A containing chromatin / 2-micrometer circle DNA / 2-micrometer plasmid partitioning / CENP-A containing nucleosome / centromeric DNA binding / kinetochore assembly / condensed chromosome, centromeric region / kinetochore => GO:0000776 / mitotic sister chromatid segregation / kinetochore / nucleosome / sequence-specific DNA binding / protein heterodimerization activity / DNA repair / DNA binding / nucleus
Similarity search - Function
Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / C-terminus of histone H2A / Histone H2A, C-terminal domain / Histone 2A ...Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / C-terminus of histone H2A / Histone H2A, C-terminal domain / Histone 2A / Histone H2A / Histone H4 signature. / Histone H4, conserved site / 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 / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA (> 100) / DNA (> 10) / DNA / Histone H3-like centromeric protein CSE4 / Histone H2A / Histone H2B.1 / Histone H4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Kluyveromyces lactis (yeast)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsMigl, D. / Kschonsak, M. / Arthur, C.P. / Khin, Y. / Harrison, S.C. / Ciferri, C. / Dimitrova, Y.N.
Funding support United States, 2items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Feb 12, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Mar 18, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Histone H3-like centromeric protein CSE4
B: Histone H4
C: Histone H2A
D: Histone H2B.1
E: Histone H3-like centromeric protein CSE4
F: Histone H4
G: Histone H2A
H: Histone H2B.1
I: DNA (119-MER)
J: DNA (119-MER)


Theoretical massNumber of molelcules
Total (without water)235,10110
Polymers235,10110
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48800 Å2
ΔGint-362 kcal/mol
Surface area69440 Å2

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3-like centromeric protein CSE4 / CENP-A homolog / Chromosome segregation protein 4


Mass: 26885.434 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CSE4, CSL2, YKL049C, YKL262 / Production host: Escherichia coli (E. coli) / References: UniProt: P36012
#2: Protein Histone H4 /


Mass: 13332.434 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) 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
Gene: KLLA0_E08647g, KLLA0_E17601g / Production host: Escherichia coli (E. coli) / References: UniProt: Q6CMU6
#3: Protein Histone H2A /


Mass: 15739.005 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) 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
Gene: HTA1, KLLA0E17413g, HTA2, KLLA0F13332g / Production host: Escherichia coli (E. coli) / References: UniProt: Q6CK59
#4: Protein Histone H2B.1


Mass: 16219.361 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) 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
Gene: HTB1, KLLA0F13310g / Production host: Escherichia coli (E. coli) / References: UniProt: Q6CK60

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (119-MER)


Mass: 45138.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain DNA (119-MER)


Mass: 45610.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Yeast Centromeric NucleosomeCOMPLEX#1-#60RECOMBINANT
2Histone H3-like centromeric protein CSE4#11RECOMBINANT
3Histone H4, Histone H2A, Histone H2B.1#2-#41RECOMBINANT
4DNA#5-#61RECOMBINANT
Molecular weightValue: 240 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
23Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)284590
34unidentified (others)32644
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.249 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 265380 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00811345
ELECTRON MICROSCOPYf_angle_d0.69616342
ELECTRON MICROSCOPYf_dihedral_angle_d21.8766003
ELECTRON MICROSCOPYf_chiral_restr0.0441875
ELECTRON MICROSCOPYf_plane_restr0.0051234

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