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- PDB-6qlf: Structure of inner kinetochore CCAN complex with mask1 -

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Entry
Database: PDB / ID: 6qlf
TitleStructure of inner kinetochore CCAN complex with mask1
Components(Inner kinetochore subunit ...) x 8
KeywordsDNA BINDING PROTEIN / inner kinetochore / CCAN
Function / homology
Function and homology information


negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / Mis6-Sim4 complex / COMA complex / establishment of meiotic sister chromatid cohesion / maintenance of meiotic sister chromatid cohesion / centromere complex assembly / meiotic sister chromatid segregation / attachment of spindle microtubules to kinetochore / ascospore formation / establishment of mitotic sister chromatid cohesion ...negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / Mis6-Sim4 complex / COMA complex / establishment of meiotic sister chromatid cohesion / maintenance of meiotic sister chromatid cohesion / centromere complex assembly / meiotic sister chromatid segregation / attachment of spindle microtubules to kinetochore / ascospore formation / establishment of mitotic sister chromatid cohesion / condensed nuclear chromosome outer kinetochore / protein localization to chromosome, centromeric region / kinetochore assembly / condensed nuclear chromosome kinetochore / spindle pole body / protein localization to kinetochore / condensed chromosome kinetochore / mitotic spindle assembly checkpoint / kinetochore / chromosome segregation / meiotic cell cycle / cell division / structural molecule activity / nucleus / cytoplasm
Centromere protein Chl4/mis15/CENP-N / Centromere protein O / Cenp-O kinetochore centromere component / Cnl2/NKP2 family protein / Kinetochore protein CHL4 like / Kinetochore subunit Nkp2/Cnl2
Inner kinetochore subunit IML3 / Inner kinetochore subunit AME1 / Inner kinetochore subunit CHL4 / Inner kinetochore subunit OKP1 / Inner kinetochore subunit CTF19 / Inner kinetochore subunit NKP2 / Inner kinetochore subunit MCM21 / Inner kinetochore subunit NKP1
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsYan, K. / Yang, J. / Zhang, Z. / McLaughlin, S.H. / Chang, L. / Fasci, D. / Heck, A.J.R. / Barford, D.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
European CommissionINFRAIA project Epic-XS (Project 823839)
CitationJournal: Nature / Year: 2019
Title: Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.
Authors: Kaige Yan / Jing Yang / Ziguo Zhang / Stephen H McLaughlin / Leifu Chang / Domenico Fasci / Ann E Ehrenhofer-Murray / Albert J R Heck / David Barford /
Abstract: In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized ...In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes, function to connect centromeric chromatin to microtubules of the mitotic spindle. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-A), each of which is perfectly centred on its cognate centromere. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-A). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-A to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-A are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-A. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJan 31, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release

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Assembly

Deposited unit
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
U: Inner kinetochore subunit AME1
Y: Inner kinetochore subunit NKP1
Z: Inner kinetochore subunit NKP2


Theoretical massNumber of molelcules
Total (without water)296,8948
Polymers296,8948
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43240 Å2
ΔGint-289 kcal/mol
Surface area83150 Å2
MethodPISA

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Components

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Inner kinetochore subunit ... , 8 types, 8 molecules LNOPQUYZ

#1: Protein/peptide Inner kinetochore subunit IML3 / 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 (baker's yeast)
Gene: IML3, MCM19, YBR107C, YBR0836 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38265
#2: Protein/peptide Inner kinetochore subunit CHL4 / CENP-N homolog / Chromosome loss protein 4 / Chromosome transmission fidelity protein 17 / Constitutive centromere-associated network protein CHL4 / Minichromosome maintenance protein 17


Mass: 53538.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CHL4, CTF17, MCM17, YDR254W, YD9320A.04 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38907
#3: Protein/peptide Inner kinetochore subunit MCM21 / 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 (baker's yeast)
Gene: MCM21, CTF5, YDR318W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q06675
#4: Protein/peptide Inner kinetochore subunit CTF19 / 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 (baker's yeast)
Gene: CTF19, MCM18, YPL018W, LPB13W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q02732
#5: Protein/peptide 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 (baker's yeast)
Gene: OKP1, YGR179C / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53298
#6: Protein/peptide Inner kinetochore subunit AME1 / Associated with microtubules and essential protein 1 / CENP-U homolog / Constitutive centromere-associated network protein AME1


Mass: 37081.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: AME1, ARP100, YBR211C, YBR1458 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38313
#7: Protein/peptide 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 (baker's yeast)
Gene: NKP1, YDR383C / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12493
#8: Protein/peptide 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 (baker's yeast)
Gene: NKP2, YLR315W / Cell line (production host): High Five / 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: Inner kinetochore CCAN complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 465029 / Symmetry type: POINT

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