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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 / 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 / Iron uptake and transport / 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 / Antigen processing: Ubiquitination & Proteasome degradation / Transcriptional activator of glycolytic enzymes / Orc1 removal from chromatin / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Neddylation / RAVE complex / CBF3 complex / septin ring assembly / centromeric DNA binding / mitotic spindle elongation / negative regulation of cytoplasmic translation / exit from mitosis / vacuolar acidification / kinetochore assembly / condensed nuclear chromosome kinetochore / protein neddylation / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / regulation of exit from mitosis / SCF ubiquitin ligase complex / spindle pole body / DNA binding, bending / DNA replication origin binding / cellular protein-containing complex assembly / condensed nuclear chromosome, centromeric region / mitotic spindle assembly checkpoint / condensed chromosome kinetochore / regulation of protein complex assembly / spindle midzone / kinetochore / chromosome segregation / G1/S transition of mitotic cell cycle / spindle / 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 / 4.2 Å resolution
AuthorsZhang, W.J. / Lukoynova, N. / Vaughan, C.K.
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 Lukoynova / 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 13, 2018 / Release: Aug 1, 2018

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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
Polyers286,5265
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
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)13640
ΔGint (kcal/M)-88
Surface area (Å2)66140
MethodPISA

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Components

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


Mass: 68454.125 Da / Num. of mol.: 2
Details: N-terminal polyhistidine purification tagTruncation of the binuclear zinc cluster domain
Mutation: Truncation of the N-terminal domain, UNP residues 1-46
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / Plasmid name: Modified pRS426 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P40969
#2: Protein/peptide 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: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: SKP1, CBF3D, YDR328C, D9798.14 / Plasmid name: Modified pRS426 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P52286
#3: Protein/peptide Centromere DNA-binding protein complex CBF3 subunit C / Chromosome transmission fidelity protein 13 / Kinetochore protein CTF13


Mass: 60899.961 Da / Num. of mol.: 1 / Details: C-terminal CBP purification tag
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CTF13, CBF3C, YMR094W, YM6543.01, YM9582.19 / Plasmid name: Modified pRS424 / Cell line (production host): BCY123 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P35203
#4: Protein/peptide 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
Details: Domains 1-2 of Ndc10 with a non-cleavable C-terminal StrepII tag
Fragment: UNP residues 2-553
Mutation: Construct comprising residues 1-554 with C-terminal Strep tag
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CBF2, CBF3A, CEP2, CTF14, NDC10, YGR140W / Plasmid name: Modified pRS426 / Cell (production host): BCY123 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32504

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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 heterodimer of Skp1 and Ctf13, and a monomeric construct Ndc10 comprising domains 1-2.
Entity ID: 1, 2, 3, 4 / Source: RECOMBINANT
Molecular weightValue: 0.286 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast) / Plasmid: modified pRS424 and pRS426
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
1200 mMsodium chlorideNaCl1
215 mMTris base1
32 mMDTT1
SpecimenConc.: 0.15 mg/ml
Details: The sample is homogeneous and well-dispersed on grids.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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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 / Calibrated magnification: 47170 / 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) / Number of grids imaged: 1 / Number 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
5CTFFIND4.0CTF correctionCTF parameters were estimated using CTFFIND4
8UCSF Chimera1.8.1model fitting
13RELION2.0initial Euler assignment
15RELION2.0final Euler assignment
17RELION2.0classification
19RELION2.03D reconstruction
Image processingDetails: The selected images were high-pass filtered and normalized.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Number of particles after selection by 2D classification
Number of particles selected: 214608
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 56509 / Algorithm: FOURIER SPACE / Number 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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