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- PDB-6gys: Cryo-EM structure of the CBF3-CEN3 complex of the budding yeast k... -

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Basic information

Entry
Database: PDB / ID: 6gys
TitleCryo-EM structure of the CBF3-CEN3 complex of the budding yeast kinetochore
Components
  • (Centromere DNA-binding protein complex CBF3 subunit ...) x 3
  • (DNA (52-MER)) x 2
  • Suppressor of kinetochore protein 1
KeywordsDNA BINDING PROTEIN / Complex
Function / homologyS-phase kinase-associated protein 1-like / Skp1 family, dimerisation domain / 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 / Ndc10, domain 2 ...S-phase kinase-associated protein 1-like / Skp1 family, dimerisation domain / 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 / Ndc10, domain 2 / Leucine-rich repeat domain superfamily / SKP1-like, dimerisation domain superfamily / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Ndc10, domain 2 superfamily / Zn(2)-C6 fungal-type DNA-binding domain / Fungal Zn(2)-Cys(6) binuclear cluster domain / Skp1 family, tetramerisation domain / Antigen processing: Ubiquitination & Proteasome degradation / Centromere DNA-binding protein complex CBF3 subunit, domain 2 / Centromere DNA-binding protein complex CBF3 subunit B / Zn(2)-C6 fungal-type DNA-binding domain signature. / Zn(2)-C6 fungal-type DNA-binding domain profile. / SCF-beta-TrCP mediated degradation of Emi1 / SCF(Skp2)-mediated degradation of p27/p21 / Transcriptional activator of glycolytic enzymes / Orc1 removal from chromatin / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Neddylation / Iron uptake and transport / RAVE complex / CBF3 complex / centromeric DNA binding / mitotic spindle elongation / septin ring assembly / negative regulation of cytoplasmic translation / vacuolar acidification / kinetochore assembly / condensed nuclear chromosome kinetochore / protein neddylation / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / regulation of exit from mitosis / exit from mitosis / SCF ubiquitin ligase complex / spindle pole body / DNA binding, bending / DNA replication origin binding / condensed nuclear chromosome, centromeric region / cellular protein-containing complex assembly / mitotic spindle assembly checkpoint / condensed chromosome kinetochore / regulation of protein complex assembly / spindle midzone / kinetochore / chromosome segregation / spindle / G1/S transition of mitotic cell cycle / ubiquitin-dependent protein catabolic process / G2/M transition of mitotic cell cycle / protein ubiquitination / DNA-binding transcription factor activity, RNA polymerase II-specific / zinc ion binding / identical protein binding / nucleus / cytoplasm / 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
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.4 Å resolution
AuthorsYan, K. / Zhang, Z. / Yang, J. / McLaughlin, S.H. / Barford, D.
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 1, 2018 / Release: Dec 5, 2018

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Assembly

Deposited unit
A: Centromere DNA-binding protein complex CBF3 subunit C
B: Centromere DNA-binding protein complex CBF3 subunit B
C: Centromere DNA-binding protein complex CBF3 subunit B
D: Suppressor of kinetochore protein 1
E: Centromere DNA-binding protein complex CBF3 subunit A
F: DNA (52-MER)
G: DNA (52-MER)
H: Centromere DNA-binding protein complex CBF3 subunit C
I: Centromere DNA-binding protein complex CBF3 subunit B
J: Centromere DNA-binding protein complex CBF3 subunit B
K: Suppressor of kinetochore protein 1
L: Centromere DNA-binding protein complex CBF3 subunit A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)699,98820
Polyers699,46512
Non-polymers5238
Water0
1


  • idetical with deposited unit
  • defined by software
  • Evidence: assay for oligomerization, SEC-MALS
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TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)48930
ΔGint (kcal/M)-254
Surface area (Å2)196180
MethodPISA

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Components

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Centromere DNA-binding protein complex CBF3 subunit ... , 3 types, 8 molecules AHBCIJEL

#1: Protein/peptide Centromere DNA-binding protein complex CBF3 subunit C / Chromosome transmission fidelity protein 13 / Kinetochore protein CTF13


Mass: 56416.863 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CTF13, CBF3C, YMR094W, YM6543.01, YM9582.19 / Production host: unidentified baculovirus / References: UniProt: P35203
#2: Protein/peptide
Centromere DNA-binding protein complex CBF3 subunit B / Centromere protein 3


Mass: 71439.891 Da / Num. of mol.: 4 / Details: Zn
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / Production host: unidentified baculovirus / References: UniProt: P40969
#4: Protein/peptide 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.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CBF2, CBF3A, CEP2, CTF14, NDC10, YGR140W / Production host: unidentified baculovirus / References: UniProt: P32504

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DNA chain , 2 types, 2 molecules FG

#5: DNA chain DNA (52-MER)


Mass: 16168.496 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: unidentified baculovirus
#6: DNA chain DNA (52-MER)


Mass: 15856.242 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: unidentified baculovirus

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Protein/peptide / Non-polymers , 2 types, 10 molecules DK

#3: Protein/peptide 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.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: SKP1, CBF3D, YDR328C, D9798.14 / Production host: unidentified baculovirus / References: UniProt: P52286
#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Formula: Zn
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: unidentified baculovirus / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction
Crystal symmetryAngle gamma: 9 deg. / Length a: 1 Å / Length b: 1 Å / Length c: 1 Å / Space group name H-M: P 1

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Sample preparation

ComponentName: Cryo-EM structure of the CBF3-CEN3 complex of the budding yeast kinetochore
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (baker's 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
MicroscopyMicroscope model: 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

Classification: refinement / Contact author: Paul D. Adams / Contact author email: pdadams[at]lbl.gov / Location: https://www.phenix-online.org/ / Type: program / Version: 1.13_2998

NameLanguage
phenix.real_space_refinePython
PHENIXC++
Crystal symmetryAngle gamma: 9 deg. / Length a: 1 Å / Length b: 1 Å / Length c: 1 Å / Space group name H-M: P 1
EM 3D crystal entityAngle alpha: 9 deg. / Angle beta: 9 deg. / Angle gamma: 9 deg. / Length a: 1 Å / Length b: 1 Å / Length c: 1 Å / Space group name: P 1 / Space group num: 1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 22668 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 6GYP
Least-squares processHighest resolution: 4.4 Å
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008040020
ELECTRON MICROSCOPYf_angle_d1.064954608
ELECTRON MICROSCOPYf_chiral_restr0.05786020
ELECTRON MICROSCOPYf_plane_restr0.00736546
ELECTRON MICROSCOPYf_dihedral_angle_d13.026823708

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