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- PDB-6fe8: Cryo-EM structure of the core Centromere Binding Factor 3 complex -

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Basic information

Entry
Database: PDB / ID: 6fe8
TitleCryo-EM structure of the core Centromere Binding Factor 3 complex
Components
  • 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 / homologyCentromere DNA-binding protein complex CBF3 subunit B / S-phase kinase-associated protein 1 / Zn(2)-C6 fungal-type DNA-binding domain profile. / Zn(2)-C6 fungal-type DNA-binding domain signature. / Zn(2)-C6 fungal-type DNA-binding domain / S-phase kinase-associated protein 1-like / SKP1/BTB/POZ domain superfamily / SCF(Skp2)-mediated degradation of p27/p21 / SKP1 component, dimerisation / SKP1 component, POZ domain ...Centromere DNA-binding protein complex CBF3 subunit B / S-phase kinase-associated protein 1 / Zn(2)-C6 fungal-type DNA-binding domain profile. / Zn(2)-C6 fungal-type DNA-binding domain signature. / Zn(2)-C6 fungal-type DNA-binding domain / S-phase kinase-associated protein 1-like / SKP1/BTB/POZ domain superfamily / SCF(Skp2)-mediated degradation of p27/p21 / SKP1 component, dimerisation / SKP1 component, POZ domain / Orc1 removal from chromatin / Centromere DNA-binding protein complex CBF3 subunit B, C-terminal / Leucine-rich repeat domain superfamily / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Neddylation / Iron uptake and transport / SKP1-like, dimerisation domain superfamily / Antigen processing: Ubiquitination & Proteasome degradation / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Fungal Zn(2)-Cys(6) binuclear cluster domain / Skp1 family, dimerisation domain / Skp1 family, tetramerisation domain / SCF-beta-TrCP mediated degradation of Emi1 / RAVE complex / CBF3 complex / septin ring assembly / centromeric DNA binding / 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 / DNA binding, bending / DNA replication origin binding / cellular protein-containing complex assembly / mitotic spindle assembly checkpoint / condensed chromosome kinetochore / regulation of protein complex assembly / kinetochore / 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 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 / 4.1 Å resolution
AuthorsZhang, W.J. / Lukoynova, N. / Miah, S. / 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: Dec 30, 2017 / Release: Aug 1, 2018
RevisionDateData content typeGroupCategoryProviderType
1.0Aug 1, 2018Structure modelrepositoryInitial release
1.1Oct 3, 2018Structure modelData collection / Refinement descriptionrefine

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-4241
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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


Theoretical massNumber of molelcules
Total (without water)220,3674
Polyers220,3674
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, It shows a single and symmetric peak at an elution volume corresponding to 220kDa.
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)13630
ΔGint (kcal/M)-88
Surface area (Å2)66160
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: Model is numbered according to
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CEP3, CBF3, CBF3B, CSL1, YMR168C, YM8520.17C / 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 / 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
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: CTF13, CBF3C, YMR094W, YM6543.01, YM9582.19 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P35203

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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: The core Centromere Binding Factor 3 complex / Type: COMPLEX
Details: The core CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, and full length Skp1 and Ctf13 components.
Entity ID: 1, 2, 3 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
1200 mMsodium chlorideNaCl1
215 mMTris base1
32 mMDTT1
SpecimenConc.: 0.12 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 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 recording

Imaging ID: 1 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1

IDAverage exposure timeElectron doseNumber of real images
10.44.61236
20.3754.061101
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.13_2992: / Classification: refinement
EM software
IDNameVersionCategoryDetailsImage processing ID
1Gautomatchparticle selectionGautomatch was used to pick a subset of the initial dataset for making references and for further automatic particle picking of the initial dataset1
3EPU1.9.1image acquisitionData collection
5CTFFIND4.0CTF correctionCTF parameters were estimated using CTFFIND41
8UCSF Chimera1.8.1model fittingUsed for initial placement of Cep3 and BTB/POZ domain of Skp1
11Coot0.8.8model fittingUsed for ab initio building of all of Ctf13, the C-terminal section of Skp1, and parts of Cep3 where new density was visible or where it deviated from the crystal structure coordinates
12PHENIX1.13model refinementReal Space Refinement Protocol
13REFMACmodel refinement
14RELION2.0initial Euler assignment1
16RELION2.0final Euler assignment1
18RELION2.0classification1
20RELION2.03D reconstruction1
22Gautomatchparticle selectionGautomatch was used to pick a subset of the initial dataset for making references and for further automatic particle picking of the initial dataset2
33CTFFIND4.0CTF correctionCTF parameters were estimated using CTFFIND42
35RELION2.0initial Euler assignment2
37RELION2.0final Euler assignment2
39RELION2.0classification2
41RELION2.03D reconstruction2
Image processing
IDImage recording IDDetails
11The selected images were high-pass filtered and normalized.
22The selected images were high-pass filtered and normalized.
CTF correction
IDEM image processing IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing IDNumber of particles selected
11139303
22289281
Symmetry
IDImage processing IDEntry IDPoint symmetry
116FE8C1
226FE8C1
3D reconstruction

Algorithm: FOURIER SPACE / Entry ID: 6FE8 / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDResolutionNumber of particlesImage processing IDDetailsNumber of class averages
14.16939213D auto-refinement of all the good particles from 2D classification, provided a reconstruction at 4.7 angstrom overall resolution. After post-process by RELION and sharpened by a negative B-factor using an automated procedure resulting in a 4.1 angstrom reconstruction.4
23.71876062187, 606 particles were in the best two 3D classes. 3D auto-refinement of the 3D joined classes against the corresponding particles resulted in a final reconstruction.2
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-IDPdb chain ID 3D fitting IDPdb chain residue range
12VEQA148-608
21NEXA1
RefineCorrelation coeff Fo to Fc: 0.884 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS / Overall ESU R: 1.068
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Solvent computationSolvent ion probe radii: 0.8 Å / Solvent shrinkage radii: 0.8 Å / Solvent vdw probe radii: 1.2 Å / Solvent model details: MASK
Displacement parametersB iso mean: 117.316 Å2 / Aniso B11: 0.92 Å2 / Aniso B12: 1.76 Å2 / Aniso B13: -5.08 Å2 / Aniso B22: -2.74 Å2 / Aniso B23: -1.5 Å2 / Aniso B33: 1.82 Å2
Least-squares processR factor R work: 0.29358 / R factor obs: 0.29358 / Highest resolution: 3.7 Å / Lowest resolution: 127.2 Å / Number reflection obs: 62402 / Percent reflection obs: 1
Number of atoms included #1Total: 12393
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0200.01912682
ELECTRON MICROSCOPYr_bond_other_d0.0000.02011685
ELECTRON MICROSCOPYr_angle_refined_deg2.2501.95517218
ELECTRON MICROSCOPYr_angle_other_deg3.7653.00026900
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.6055.0001535
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.62523.657566
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.12615.0002140
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.56315.00068
ELECTRON MICROSCOPYr_chiral_restr0.1490.2001966
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.02013935
ELECTRON MICROSCOPYr_gen_planes_other0.0130.0202754
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it18.68910.9986176
ELECTRON MICROSCOPYr_mcbond_other18.68310.9976175
ELECTRON MICROSCOPYr_mcangle_it30.38616.3937699
ELECTRON MICROSCOPYr_mcangle_other30.38416.3957700
ELECTRON MICROSCOPYr_scbond_it26.28913.1336506
ELECTRON MICROSCOPYr_scbond_other26.28713.1336506
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other41.65818.7279520
ELECTRON MICROSCOPYr_long_range_B_refined50.88614953
ELECTRON MICROSCOPYr_long_range_B_other50.84614949
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 3.7 Å / R factor R work: 0.615 / Lowest resolution: 3.796 Å / Number reflection R free: 0 / Number reflection R work: 4633 / Total number of bins used: 20 / Percent reflection obs: 1

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