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- PDB-6gyu: Cryo-EM structure of the CBF3-msk complex of the budding yeast ki... -

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Basic information

Entry
Database: PDB / ID: 6gyu
TitleCryo-EM structure of the CBF3-msk complex of the budding yeast kinetochore
Components
  • (Centromere DNA-binding protein complex CBF3 subunit ...) x 4
  • Suppressor of kinetochore protein 1
KeywordsDNA BINDING PROTEIN / Complex
Function / homology
Function and homology information


RAVE complex / CBF3 complex / septin ring assembly / mitotic spindle elongation / negative regulation of cytoplasmic translation / centromeric DNA binding / vacuolar acidification / exit from mitosis / protein neddylation / kinetochore assembly ...RAVE complex / CBF3 complex / septin ring assembly / mitotic spindle elongation / negative regulation of cytoplasmic translation / centromeric DNA binding / vacuolar acidification / exit from mitosis / protein neddylation / kinetochore assembly / regulation of exit from mitosis / condensed nuclear chromosome kinetochore / SCF ubiquitin ligase complex / spindle pole body / DNA binding, bending / cullin family protein binding / DNA replication origin binding / condensed nuclear chromosome, centromeric region / cellular protein-containing complex assembly / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / condensed chromosome kinetochore / mitotic spindle assembly checkpoint / regulation of protein-containing complex assembly / spindle midzone / kinetochore / chromosome segregation / spindle / G1/S transition of mitotic cell cycle / mitotic cell cycle / G2/M transition of mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein ubiquitination / DNA-binding transcription factor activity, RNA polymerase II-specific / cell / zinc ion binding / identical protein binding / nucleus / cytoplasm
Centromere DNA-binding protein complex CBF3 subunit, domain 2 / Ndc10, domain 2 / Zn(2)-C6 fungal-type DNA-binding domain / S-phase kinase-associated protein 1-like / Integrase/recombinase, N-terminal / SKP1/BTB/POZ domain superfamily / SKP1 component, dimerisation / SKP1 component, POZ domain / S-phase kinase-associated protein 1 / Transcription activator GCR1-like domain ...Centromere DNA-binding protein complex CBF3 subunit, domain 2 / Ndc10, domain 2 / Zn(2)-C6 fungal-type DNA-binding domain / S-phase kinase-associated protein 1-like / Integrase/recombinase, N-terminal / SKP1/BTB/POZ domain superfamily / SKP1 component, dimerisation / SKP1 component, POZ domain / S-phase kinase-associated protein 1 / Transcription activator GCR1-like domain / Centromere DNA-binding protein complex CBF3 subunit B, C-terminal / Leucine-rich repeat domain superfamily / SKP1-like, dimerisation domain superfamily / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Ndc10, domain 2 superfamily / Fungal Zn(2)-Cys(6) binuclear cluster domain / Centromere DNA-binding protein complex CBF3 subunit B / Skp1 family, dimerisation domain / Skp1 family, tetramerisation domain / Transcriptional activator of glycolytic enzymes
Centromere DNA-binding protein complex CBF3 subunit A / Centromere DNA-binding protein complex CBF3 subunit C / Centromere DNA-binding protein complex CBF3 subunit B / Suppressor of kinetochore protein 1
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsYan, K. / Zhang, Z. / Yang, J. / McLaughlin, S.H. / Barford, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MRC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: Architecture of the CBF3-centromere complex of the budding yeast kinetochore.
Authors: Kaige Yan / Ziguo Zhang / Jing Yang / Stephen H McLaughlin / David Barford /
Abstract: Kinetochores are multicomponent complexes responsible for coordinating the attachment of centromeric DNA to mitotic-spindle microtubules. The point centromeres of budding yeast are organized into ...Kinetochores are multicomponent complexes responsible for coordinating the attachment of centromeric DNA to mitotic-spindle microtubules. The point centromeres of budding yeast are organized into three centromeric determining elements (CDEs), and are associated with the centromere-specific nucleosome Cse4. Deposition of Cse4 at CEN loci is dependent on the CBF3 complex that engages CDEIII to direct Cse4 nucleosomes to CDEII. To understand how CBF3 recognizes CDEIII and positions Cse4, we determined a cryo-EM structure of a CBF3-CEN complex. CBF3 interacts with CEN DNA as a head-to-head dimer that includes the whole of CDEIII and immediate 3' regions. Specific CEN-binding of CBF3 is mediated by a Cep3 subunit of one of the CBF3 protomers that forms major groove interactions with the conserved and essential CCG and TGT motifs of CDEIII. We propose a model for a CBF3-Cse4-CEN complex with implications for understanding CBF3-directed deposition of the Cse4 nucleosome at CEN loci.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 5, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
B: Centromere DNA-binding protein complex CBF3 subunit B
A: Centromere DNA-binding protein complex CBF3 subunit C
C: Centromere DNA-binding protein complex CBF3 subunit B
D: Suppressor of kinetochore protein 1
E: Centromere DNA-binding protein complex CBF3 subunit A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)328,8137
Polymers328,6825
Non-polymers1312
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19480 Å2
ΔGint-90 kcal/mol
Surface area90860 Å2
MethodPISA

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Components

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Centromere DNA-binding protein complex CBF3 subunit ... , 4 types, 4 molecules BACE

#1: Protein Centromere DNA-binding protein complex CBF3 subunit B / Centromere protein 3


Mass: 71439.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / Production host: unidentified baculovirus / References: UniProt: P40969
#2: Protein Centromere DNA-binding protein complex CBF3 subunit C / Chromosome transmission fidelity protein 13 / Kinetochore protein CTF13


Mass: 56416.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CTF13, CBF3C, YMR094W, YM6543.01, YM9582.19 / Production host: unidentified baculovirus / References: UniProt: P35203
#3: Protein Centromere DNA-binding protein complex CBF3 subunit B / Centromere protein 3


Mass: 66401.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / Production host: unidentified baculovirus / References: UniProt: P40969
#5: Protein Centromere DNA-binding protein complex CBF3 subunit A / Centromere-binding factor 2 / Chromosome transmission fidelity protein 14 / Kinetochore protein CTF14


Mass: 112066.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CBF2, CBF3A, CEP2, CTF14, NDC10, YGR140W / Production host: unidentified baculovirus / References: UniProt: P32504

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Protein / Non-polymers , 2 types, 3 molecules D

#4: Protein Suppressor of kinetochore protein 1 / Centromere DNA-binding protein complex CBF3 subunit D / E3 ubiquitin ligase complex SCF subunit SKP1


Mass: 22357.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SKP1, CBF3D, YDR328C, D9798.14 / Production host: unidentified baculovirus / References: UniProt: P52286
#6: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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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: CBF3-msk complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: unidentified baculovirus
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 27 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
PHENIX1.13_2998refinement
PHENIX1.13_2998refinement
PHENIX1.13_2998refinement
PHENIX1.13_2998refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198010 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6GYP
RefinementHighest resolution: 3 Å
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005118597
ELECTRON MICROSCOPYf_angle_d0.872325179
ELECTRON MICROSCOPYf_chiral_restr0.05312772
ELECTRON MICROSCOPYf_plane_restr0.00693186
ELECTRON MICROSCOPYf_dihedral_angle_d9.904611185

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