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- PDB-6gsa: Core Centromere Binding Factor 3 (CBF3) with monomeric Ndc10 -

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Basic information

Entry
Database: PDB / ID: 6gsa
TitleCore Centromere Binding Factor 3 (CBF3) with monomeric Ndc10
Components
  • Centromere DNA-binding protein complex CBF3 subunit A
  • Centromere DNA-binding protein complex CBF3 subunit B
  • Centromere DNA-binding protein complex CBF3 subunit C
  • Suppressor of kinetochore protein 1
KeywordsDNA BINDING PROTEIN / Centromere / CDEIII-binding / LRR domain
Function / homology
Function and homology information


RAVE complex / Iron uptake and transport / CBF3 complex / regulation of transcription by galactose / regulation of sulfur amino acid metabolic process / cellular response to methylmercury / vacuolar proton-transporting V-type ATPase complex assembly / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / septin ring assembly / mitotic spindle elongation ...RAVE complex / Iron uptake and transport / CBF3 complex / regulation of transcription by galactose / regulation of sulfur amino acid metabolic process / cellular response to methylmercury / vacuolar proton-transporting V-type ATPase complex assembly / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / septin ring assembly / mitotic spindle elongation / centromeric DNA binding / regulation of exit from mitosis / Antigen processing: Ubiquitination & Proteasome degradation / vacuolar acidification / kinetochore assembly / condensed chromosome, centromeric region / regulation of metabolic process / exit from mitosis / spindle pole body / positive regulation of glucose transmembrane transport / protein neddylation / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / mitochondrial fusion / DNA binding, bending / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / SCF ubiquitin ligase complex / mitotic spindle assembly checkpoint signaling / DNA replication origin binding / regulation of mitotic cell cycle / cullin family protein binding / subtelomeric heterochromatin formation / regulation of protein-containing complex assembly / spindle midzone / endomembrane system / negative regulation of cytoplasmic translation / chromosome segregation / G1/S transition of mitotic cell cycle / spindle / kinetochore / G2/M transition of mitotic cell cycle / mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein-containing complex assembly / chromosome, telomeric region / protein ubiquitination / DNA-binding transcription factor activity, RNA polymerase II-specific / DNA binding / zinc ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
: / Ndc10 N-terminal domain / Transcription activator GCR1-like domain / Ndc10, domain 2 / Ndc10, domain 2 superfamily / Transcriptional activator of glycolytic enzymes / Centromere DNA-binding protein complex CBF3 subunit, domain 2 / Centromere DNA-binding protein complex CBF3 subunit B, C-terminal / Centromere DNA-binding protein complex CBF3 subunit B / Zn(2)-C6 fungal-type DNA-binding domain signature. ...: / Ndc10 N-terminal domain / Transcription activator GCR1-like domain / Ndc10, domain 2 / Ndc10, domain 2 superfamily / Transcriptional activator of glycolytic enzymes / Centromere DNA-binding protein complex CBF3 subunit, domain 2 / Centromere DNA-binding protein complex CBF3 subunit B, C-terminal / Centromere DNA-binding protein complex CBF3 subunit B / Zn(2)-C6 fungal-type DNA-binding domain signature. / Fungal Zn(2)-Cys(6) binuclear cluster domain / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Zn(2)-C6 fungal-type DNA-binding domain profile. / GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA-binding domain / Zn(2)-C6 fungal-type DNA-binding domain / Integrase/recombinase, N-terminal / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
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
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsZhang, W.J. / Lukoynova, N. / Vaughan, C.K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/J007595/1 United Kingdom
CitationJournal: Cell Rep / Year: 2018
Title: Insights into Centromere DNA Bending Revealed by the Cryo-EM Structure of the Core Centromere Binding Factor 3 with Ndc10.
Authors: Wenjuan Zhang / Natalya Lukoyanova / Shomon Miah / Jonathan Lucas / Cara K Vaughan /
Abstract: The centromere binding factor 3 (CBF3) complex binds the third centromere DNA element in organisms with point centromeres, such as S. cerevisiae. It is an essential complex for assembly of the ...The centromere binding factor 3 (CBF3) complex binds the third centromere DNA element in organisms with point centromeres, such as S. cerevisiae. It is an essential complex for assembly of the kinetochore in these organisms, as it facilitates genetic centromere specification and allows association of all other kinetochore components. We determined high-resolution structures of the core complex of CBF3 alone and in association with a monomeric construct of Ndc10, using cryoelectron microscopy (cryo-EM). We identify the DNA-binding site of the complex and present a model in which CBF3 induces a tight bend in centromeric DNA, thus facilitating assembly of the centromeric nucleosome.
History
DepositionJun 13, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)286,5265
Polymers286,5265
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Complex migrates as a single peak on analytical gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13640 Å2
ΔGint-88 kcal/mol
Surface area66140 Å2
MethodPISA

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Components

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


Mass: 68454.125 Da / Num. of mol.: 2
Mutation: Truncation of the N-terminal domain, UNP residues 1-46
Source method: isolated from a genetically manipulated source
Details: N-terminal polyhistidine purification tagTruncation of the binuclear zinc cluster domain
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / Plasmid: Modified pRS426 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40969
#2: Protein Suppressor of kinetochore protein 1 / Centromere DNA-binding protein complex CBF3 subunit D / E3 ubiquitin ligase complex SCF subunit SKP1


Mass: 22558.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SKP1, CBF3D, YDR328C, D9798.14 / Plasmid: Modified pRS426 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P52286
#3: Protein Centromere DNA-binding protein complex CBF3 subunit C / Chromosome transmission fidelity protein 13 / Kinetochore protein CTF13


Mass: 60899.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal CBP purification tag
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF13, CBF3C, YMR094W, YM6543.01, YM9582.19 / Plasmid: Modified pRS424 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P35203
#4: Protein Centromere DNA-binding protein complex CBF3 subunit A / Centromere-binding factor 2 / Chromosome transmission fidelity protein 14 / Kinetochore protein CTF14


Mass: 66159.578 Da / Num. of mol.: 1 / Fragment: UNP residues 2-553
Mutation: Construct comprising residues 1-554 with C-terminal Strep tag
Source method: isolated from a genetically manipulated source
Details: Domains 1-2 of Ndc10 with a non-cleavable C-terminal StrepII tag
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CBF2, CBF3A, CEP2, CTF14, NDC10, YGR140W / Plasmid: Modified pRS426 / Cell (production host): BCY123 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32504

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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: Core CBF3 in complex with Ndc10 D1-2 / Type: COMPLEX
Details: The truncated CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, full length ...Details: The truncated CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, full length heterodimer of Skp1 and Ctf13, and a monomeric construct Ndc10 comprising domains 1-2.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.286 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: modified pRS424 and pRS426
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
215 mMTris base1
32 mMDTT1
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample is homogeneous and well-dispersed on grids.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Calibrated magnification: 47170 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2003
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
3EPU1.9.1image acquisitionData collection
5CTFFIND4CTF correctionCTF parameters were estimated using CTFFIND4
8UCSF Chimera1.8.1model fitting
13RELION2initial Euler assignment
15RELION2final Euler assignment
17RELION2classification
19RELION23D reconstruction
Image processingDetails: The selected images were high-pass filtered and normalized.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 214608
Details: Number of particles after selection by 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56509 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: "Fit in map" function used to place 6FE8 and 4ACO with out further refinement or model building

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