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- PDB-8ovw: Cryo-EM structure of CBF1-CCAN bound topologically to centromeric DNA -

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Basic information

Entry
Database: PDB / ID: 8ovw
TitleCryo-EM structure of CBF1-CCAN bound topologically to centromeric DNA
Components
  • (C0N3 DNA) x 2
  • (Inner kinetochore subunit ...) x 13
  • Centromere-binding protein 1
KeywordsCELL CYCLE / kinetochore / point centromere / CENP-A nucleosome / topological entrapment / centromeric DNA
Function / homology
Function and homology information


Cbf1-Met4-Met28 complex / regulation of sulfur metabolic process / negative regulation of kinetochore assembly / negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / maintenance of meiotic sister chromatid cohesion / meiotic sister chromatid segregation / Mis6-Sim4 complex / centromere complex assembly / establishment of meiotic sister chromatid cohesion ...Cbf1-Met4-Met28 complex / regulation of sulfur metabolic process / negative regulation of kinetochore assembly / negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / maintenance of meiotic sister chromatid cohesion / meiotic sister chromatid segregation / Mis6-Sim4 complex / centromere complex assembly / establishment of meiotic sister chromatid cohesion / ascospore formation / attachment of spindle microtubules to kinetochore / centromeric DNA binding / CENP-A containing chromatin assembly / outer kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / kinetochore assembly / spindle pole body / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / chromosome, centromeric region / DNA replication initiation / meiotic cell cycle / chromosome segregation / kinetochore / DNA-binding transcription repressor activity, RNA polymerase II-specific / DNA-binding transcription activator activity, RNA polymerase II-specific / RNA polymerase II-specific DNA-binding transcription factor binding / protein dimerization activity / chromatin remodeling / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA-binding transcription factor activity / cell division / protein-containing complex binding / regulation of transcription by RNA polymerase II / structural molecule activity / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / nucleus / cytoplasm
Similarity search - Function
: / Kinetochore subunit Nkp2/Cnl2 / Cnl2/NKP2 family protein / : / : / CENPH protein / CENPU protein / Centromere protein O / Cenp-O kinetochore centromere component / Centromere protein Chl4/mis15/CENP-N ...: / Kinetochore subunit Nkp2/Cnl2 / Cnl2/NKP2 family protein / : / : / CENPH protein / CENPU protein / Centromere protein O / Cenp-O kinetochore centromere component / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Helix-loop-helix DNA-binding domain / Helix-loop-helix DNA-binding domain superfamily / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile.
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Centromere-binding protein 1 / Inner kinetochore subunit IML3 / Inner kinetochore subunit AME1 / Inner kinetochore subunit CHL4 / Inner kinetochore subunit CNN1 / Inner kinetochore subunit MCM22 / Inner kinetochore subunit OKP1 ...DNA / DNA (> 10) / DNA (> 100) / Centromere-binding protein 1 / Inner kinetochore subunit IML3 / Inner kinetochore subunit AME1 / Inner kinetochore subunit CHL4 / Inner kinetochore subunit CNN1 / Inner kinetochore subunit MCM22 / Inner kinetochore subunit OKP1 / Inner kinetochore subunit CTF19 / Inner kinetochore subunit NKP2 / Inner kinetochore subunit MCM21 / Inner kinetochore subunit MCM16 / Inner kinetochore subunit NKP1 / Inner kinetochore subunit CTF3 / Inner kinetochore subunit WIP1
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. / Barford, D.
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

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Assembly

Deposited unit
A: Centromere-binding protein 1
B: Centromere-binding protein 1
D: C0N3 DNA
H: Inner kinetochore subunit MCM16
I: Inner kinetochore subunit CTF3
K: Inner kinetochore subunit MCM22
L: Inner kinetochore subunit IML3
N: Inner kinetochore subunit CHL4
O: Inner kinetochore subunit MCM21
P: Inner kinetochore subunit CTF19
Q: Inner kinetochore subunit OKP1
T: Inner kinetochore subunit CNN1
U: Inner kinetochore subunit AME1
W: Inner kinetochore subunit WIP1
Y: Inner kinetochore subunit NKP1
Z: Inner kinetochore subunit NKP2
E: C0N3 DNA


Theoretical massNumber of molelcules
Total (without water)654,57717
Polymers654,57717
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area98070 Å2
ΔGint-640 kcal/mol
Surface area157540 Å2
MethodPISA

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Centromere-binding protein 1 / CBP-1 / Centromere promoter factor 1 / Centromere-binding factor 1


Mass: 39444.715 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CBF1, CEP1, CP1, CPF1, YJR060W, J1730 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P17106

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DNA chain , 2 types, 2 molecules DE

#2: DNA chain C0N3 DNA


Mass: 47194.199 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#16: DNA chain C0N3 DNA


Mass: 47239.277 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Inner kinetochore subunit ... , 13 types, 13 molecules HIKLNOPQTUWYZ

#3: Protein Inner kinetochore subunit MCM16 / CENP-H homolog / Constitutive centromere-associated network protein MCM16 / Minichromosome ...CENP-H homolog / Constitutive centromere-associated network protein MCM16 / Minichromosome maintenance protein 16


Mass: 21166.100 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM16, YPR046W / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12262
#4: Protein Inner kinetochore subunit CTF3 / CENP-I homolog / Chromosome loss protein 3 / Chromosome transmission fidelity protein 3 / ...CENP-I homolog / Chromosome loss protein 3 / Chromosome transmission fidelity protein 3 / Constitutive centromere-associated network protein CTF3


Mass: 84345.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF3, CHL3, YLR381W / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12748
#5: Protein Inner kinetochore subunit MCM22 / CENP-K homolog / Constitutive centromere-associated network protein MCM22 / Minichromosome ...CENP-K homolog / Constitutive centromere-associated network protein MCM22 / Minichromosome maintenance protein 22


Mass: 27602.541 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM22, YJR135C, J2122 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P47167
#6: Protein Inner kinetochore subunit IML3 / CENP-L homolog / Constitutive centromere-associated network protein IML3 / Increased minichromosome ...CENP-L homolog / Constitutive centromere-associated network protein IML3 / Increased minichromosome loss protein 3 / Minichromosome maintenance protein 19


Mass: 28093.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: IML3, MCM19, YBR107C, YBR0836 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38265
#7: Protein Inner kinetochore subunit CHL4 / CENP-N homolog / Chromosome loss protein 4 / Chromosome transmission fidelity protein 17 / ...CENP-N homolog / Chromosome loss protein 4 / Chromosome transmission fidelity protein 17 / Constitutive centromere-associated network protein CHL4 / Minichromosome maintenance protein 17


Mass: 52743.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CHL4, CTF17, MCM17, YDR254W, YD9320A.04 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38907
#8: 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
#9: 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
#10: 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
#11: Protein Inner kinetochore subunit CNN1 / CENP-T homolog / Co-purified with NNF1 protein 1 / Constitutive centromere-associated network protein CNN1


Mass: 41359.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CNN1, YFR046C / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P43618
#12: 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
#13: Protein Inner kinetochore subunit WIP1 / CENP-W homolog / Constitutive centromere-associated network protein WIP1 / W-like protein 1


Mass: 10255.458 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: WIP1, YDR374W-A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q2V2P8
#14: 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
#15: 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: 43467 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00429063
ELECTRON MICROSCOPYf_angle_d0.75139445
ELECTRON MICROSCOPYf_dihedral_angle_d18.5894183
ELECTRON MICROSCOPYf_chiral_restr0.0454533
ELECTRON MICROSCOPYf_plane_restr0.0054819

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