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- PDB-8ovx: Cryo-EM structure of yeast CENP-OPQU+ bound to the CENP-A N-terminus -

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Basic information

Entry
Database: PDB / ID: 8ovx
TitleCryo-EM structure of yeast CENP-OPQU+ bound to the CENP-A N-terminus
Components(Inner kinetochore subunit ...) x 6
KeywordsCELL CYCLE / kinetochore / point centromere / CENP-A nucleosome / topological entrapment / centromeric DNA
Function / homology
Function and homology information


negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / Mis6-Sim4 complex / centromere complex assembly / attachment of spindle microtubules to kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / spindle pole body / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling ...negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / Mis6-Sim4 complex / centromere complex assembly / attachment of spindle microtubules to kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / spindle pole body / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / meiotic cell cycle / chromosome segregation / kinetochore / cell division / nucleus / cytoplasm
Similarity search - Function
Kinetochore subunit Nkp2/Cnl2 / Cnl2/NKP2 family protein / : / CENPU protein / Centromere protein O / Cenp-O kinetochore centromere component
Similarity search - Domain/homology
Inner kinetochore subunit AME1 / Inner kinetochore subunit OKP1 / Inner kinetochore subunit CTF19 / Inner kinetochore subunit NKP2 / Inner kinetochore subunit MCM21 / Inner kinetochore subunit NKP1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDendooven, T.D. / Zhang, Z. / Yang, J. / McLaughlin, S. / Schwabb, J. / Scheres, S. / Yatskevich, S.
Funding support United Kingdom, Germany, 4items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
UK Research and Innovation (UKRI)MC_UP_ A025_1013 United Kingdom
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.
Authors: Tom Dendooven / Ziguo Zhang / Jing Yang / Stephen H McLaughlin / Johannes Schwab / Sjors H W Scheres / Stanislau Yatskevich / David Barford /
Abstract: The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) ...The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-A). This revealed a central CENP-A with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-A can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
History
DepositionApr 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
O: Inner kinetochore subunit MCM21
P: Inner kinetochore subunit CTF19
Q: Inner kinetochore subunit OKP1
U: Inner kinetochore subunit AME1
Y: Inner kinetochore subunit NKP1
Z: Inner kinetochore subunit NKP2


Theoretical massNumber of molelcules
Total (without water)215,6876
Polymers215,6876
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23040 Å2
ΔGint-178 kcal/mol
Surface area44890 Å2
MethodPISA

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Components

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Inner kinetochore subunit ... , 6 types, 6 molecules OPQUYZ

#1: Protein Inner kinetochore subunit MCM21 / CENP-O homolog / Chromosome transmission fidelity protein 5 / Constitutive centromere-associated ...CENP-O homolog / Chromosome transmission fidelity protein 5 / Constitutive centromere-associated network protein MCM21 / Minichromosome maintenance protein 21


Mass: 43028.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM21, CTF5, YDR318W / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q06675
#2: Protein Inner kinetochore subunit CTF19 / CENP-P homolog / Chromosome transmission fidelity protein 19 / Constitutive centromere-associated ...CENP-P homolog / Chromosome transmission fidelity protein 19 / Constitutive centromere-associated network protein CTF19 / Minichromosome maintenance protein 18


Mass: 42841.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF19, MCM18, YPL018W, LPB13W / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q02732
#3: Protein Inner kinetochore subunit OKP1 / CENP-Q homolog / Constitutive centromere-associated network protein OKP1 / Outer kinetochore protein 1


Mass: 47427.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: OKP1, YGR179C / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53298
#4: Protein Inner kinetochore subunit AME1 / Associated with microtubules and essential protein 1 / CENP-U homolog / Constitutive centromere- ...Associated with microtubules and essential protein 1 / CENP-U homolog / Constitutive centromere-associated network protein AME1


Mass: 37506.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: AME1, ARP100, YBR211C, YBR1458 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38313
#5: Protein Inner kinetochore subunit NKP1 / Constitutive centromere-associated network protein NKP1 / Non-essential kinetochore protein 1


Mass: 27006.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NKP1, YDR383C / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12493
#6: Protein Inner kinetochore subunit NKP2 / Constitutive centromere-associated network protein NKP2 / Non-essential kinetochore protein 2


Mass: 17877.033 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NKP2, YLR315W / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q06162

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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

ComponentName: A complex of CBF1-CCAN bound to centromeric C0N3 DNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 595147 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046845
ELECTRON MICROSCOPYf_angle_d0.9449236
ELECTRON MICROSCOPYf_dihedral_angle_d7.892927
ELECTRON MICROSCOPYf_chiral_restr0.0531116
ELECTRON MICROSCOPYf_plane_restr0.0041159

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