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- EMDB-4241: Cryo-EM structure of the core Centromere Binding Factor 3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4241
TitleCryo-EM structure of the core Centromere Binding Factor 3 complex
Map data
Sample
  • Complex: The core Centromere Binding Factor 3 complex
    • Protein or peptide: Centromere DNA-binding protein complex CBF3 subunit B
    • Protein or peptide: Suppressor of kinetochore protein 1
    • Protein or peptide: Centromere DNA-binding protein complex CBF3 subunit C
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 / septin ring assembly / centromeric DNA binding / regulation of exit from mitosis ...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 / septin ring assembly / centromeric DNA binding / regulation of exit from mitosis / kinetochore assembly / vacuolar acidification / positive regulation of D-glucose transmembrane transport / protein neddylation / regulation of metabolic process / mitochondrial fusion / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / DNA binding, bending / exit from mitosis / SCF ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / mitotic spindle assembly checkpoint signaling / Orc1 removal from chromatin / DNA replication origin binding / cullin family protein binding / Antigen processing: Ubiquitination & Proteasome degradation / regulation of protein-containing complex assembly / subtelomeric heterochromatin formation / endomembrane system / negative regulation of cytoplasmic translation / regulation of mitotic cell cycle / kinetochore / G1/S transition of mitotic cell cycle / G2/M transition of mitotic cell cycle / mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein-containing complex assembly / chromosome, telomeric region / DNA-binding transcription factor activity, RNA polymerase II-specific / protein ubiquitination / zinc ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
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 / SKP1 component, dimerisation ...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 / 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 C / Centromere DNA-binding protein complex CBF3 subunit B / Suppressor of kinetochore protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang WJ / Lukoynova N / Miah S / Vaughan CK
Funding support United Kingdom, 1 items
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
DepositionDec 30, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseAug 1, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6fe8
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4241.png

Downloads & links

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Map

FileDownload / File: emd_4241.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212. Å		1.06 Å/pix.
x 200 pix.
= 212. Å		1.06 Å/pix.
x 200 pix.
= 212. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0664 / Movie #1: 0.0664
Minimum - Maximum-0.18915181 - 0.3365617
Average (Standard dev.)0.00065497926 (±0.0142806135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1890.3370.001

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Supplemental data

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Mask #1

Fileemd_4241_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the final map, which was used for FSC calculation.

Fileemd_4241_half_map_1.map
AnnotationHalf map of the final map, which was used for FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The other half map of the final map,...

Fileemd_4241_half_map_2.map
AnnotationThe other half map of the final map, which was used for FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The core Centromere Binding Factor 3 complex

EntireName: The core Centromere Binding Factor 3 complex
Components
  • Complex: The core Centromere Binding Factor 3 complex
    • Protein or peptide: Centromere DNA-binding protein complex CBF3 subunit B
    • Protein or peptide: Suppressor of kinetochore protein 1
    • Protein or peptide: Centromere DNA-binding protein complex CBF3 subunit C

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Supramolecule #1: The core Centromere Binding Factor 3 complex

SupramoleculeName: The core Centromere Binding Factor 3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Centromere DNA-binding protein complex CBF3 subunit B

MacromoleculeName: Centromere DNA-binding protein complex CBF3 subunit B / type: protein_or_peptide / ID: 1 / Details: Model is numbered according to / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 68.454125 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGGSSHHHHH HSSGLVPRGS HMKLITASSS KEYLPDLLLF WQNYEYWITN IGLYKTKQRD LTRTPANLDT DTEECMFWMN YLQKDQSFQ LMNFAMENLG ALYFGSIGDI SELYLRVEQY WDRRADKNHS VDGKYWDALI WSVFTMCIYY MPVEKLAEIF S VYPLHEYL ...String:
MGGSSHHHHH HSSGLVPRGS HMKLITASSS KEYLPDLLLF WQNYEYWITN IGLYKTKQRD LTRTPANLDT DTEECMFWMN YLQKDQSFQ LMNFAMENLG ALYFGSIGDI SELYLRVEQY WDRRADKNHS VDGKYWDALI WSVFTMCIYY MPVEKLAEIF S VYPLHEYL GSNKRLNWED GMQLVMCQNF ARCSLFQLKQ CDFMAHPDIR LVQAYLILAT TTFPYDEPLL ANSLLTQCIH TF KNFHVDD FRPLLNDDPV ESIAKVTLGR IFYRLCGCDY LQSGPRKPIA LHTEVSSLLQ HAAYLQDLPN VDVYREENST EVL YWKIIS LDRDLDQYLN KSSKPPLKTL DAIRRELDIF QYKVDSLEED FRSNNSRFQK FIALFQISTV SWKLFKMYLI YYDT ADSLL KVIHYSKVII SLIVNNFHAK SEFFNRHPMV MQTITRVVSF ISFYQIFVES AAVKQLLVDL TELTANLPTI FGSKL DKLV YLTERLSKLK LLWDKVQLLD SGDSFYHPVF KILQNDIKII ELKNDEMFSL IKGLGSLVPL NKLRQESLLE EEDENN TEP SDFRTIVEEF QSEYNISDIL S

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Macromolecule #2: Suppressor of kinetochore protein 1

MacromoleculeName: Suppressor of kinetochore protein 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 22.558451 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGVTSNVVLV SGEGERFTVD KKIAERSLLL KNYLNDMHDS NLQNNSDSES DSDSETNHKS KDNNNGDDDD EDDDEIVMPV PNVRSSVLQ KVIEWAEHHR DSNFPDEDDD DSRKSAPVDS WDREFLKVDQ EMLYEIILAA NYLNIKPLLD AGCKVVAEMI R GRSPEEIR ...String:
MGVTSNVVLV SGEGERFTVD KKIAERSLLL KNYLNDMHDS NLQNNSDSES DSDSETNHKS KDNNNGDDDD EDDDEIVMPV PNVRSSVLQ KVIEWAEHHR DSNFPDEDDD DSRKSAPVDS WDREFLKVDQ EMLYEIILAA NYLNIKPLLD AGCKVVAEMI R GRSPEEIR RTFNIVNDFT PEEEAAIRRE NEWAEDRGS

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Macromolecule #3: Centromere DNA-binding protein complex CBF3 subunit C

MacromoleculeName: Centromere DNA-binding protein complex CBF3 subunit C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 60.899961 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGPSFNPVRF LELPIDIRKE VYFHLDGNFC GAHPYPIDIL YKSNDVELPG KPSYKRSKRS KKLLRYMYPV FATYLNIFEY SPQLIEKWL EYAFWLRYDC LVLDCFKVNH LYDGTLIDAL EWTYLDNELR LAYFNKASML EVWYTFKEYK KWVIDSVAFD E LDLLNVSN ...String:
MGPSFNPVRF LELPIDIRKE VYFHLDGNFC GAHPYPIDIL YKSNDVELPG KPSYKRSKRS KKLLRYMYPV FATYLNIFEY SPQLIEKWL EYAFWLRYDC LVLDCFKVNH LYDGTLIDAL EWTYLDNELR LAYFNKASML EVWYTFKEYK KWVIDSVAFD E LDLLNVSN IQFNIDNLTP QLVDKCLSIL EQKDLFATIG EVQFGQDEEV GEEKDVDVSG ANSDENSSPS STIKNKKRSA SK RSHSDNG NVGATHNQLT SISVIRTIRS MESMKSLRKI TVRGEKLYEL LINFHGFRDN PGKTISYIVK RRINEIRLSR MNQ ISRTGL ADFTRWDNLQ KLVLSRVAYI DLNSIVFPKN FKSLTMKRVS KIKWWNIEEN ILKELKVDKR TFKSLYIKED DSKF TKFFN LRHTRIKELD KSEINQITYL RCQAIVWLSF RTLNHIKLQN VSEVFNNIIV PRALFDSKRV EIYRCEKISQ VLVIG SRSG SENLYFQGSK RRWKKNFIAV SAANRFKKIS SSGAL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.12 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
15.0 mMTris base
2.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsThe sample is homogeneous and well-dispersed on grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 1 / #0 - Number real images: 1236 / #0 - Average exposure time: 0.4 sec. / #0 - Average electron dose: 4.6 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Digitization - Frames/image: 1-40 / #1 - Number grids imaged: 1 / #1 - Number real images: 1101 / #1 - Average exposure time: 0.375 sec. / #1 - Average electron dose: 4.06 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47170 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Image processing ID1
Image recording ID1
DetailsThe selected images were high-pass filtered and normalized.
Particle selectionNumber selected: 139303
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Software - details: CTF parameters were estimated using CTFFIND4
Startup modelType of model: OTHER
Details: An ovoid generated from SPIDER was used as an initial model for 3D classification.
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: 3D 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- ...Details: 3D 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.
Number images used: 69392
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 17348 / Software - Name: RELION (ver. 2.0)
Details: The automatically picked particles were screened manually followed by reference-free 2D classification, which yielded 69,392 particles for subsequent 3D classification.
FSC plot (resolution estimation)

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Image processing #2

Image processing ID2
Image recording ID2
DetailsThe selected images were high-pass filtered and normalized.
Particle selectionNumber selected: 289281
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Software - details: CTF parameters were estimated using CTFFIND4
Startup modelType of model: OTHER
Details: An ovoid generated from SPIDER was used as an initial model for 3D classification.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: 187, 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.
Number images used: 187606
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 94038 / Software - Name: RELION (ver. 2.0)
Details: After joining the two dataset of selection from 2D classification , 282,116 particles were input to 3D classification using an initial 3D reference obtained by low pass-filtering the ...Details: After joining the two dataset of selection from 2D classification , 282,116 particles were input to 3D classification using an initial 3D reference obtained by low pass-filtering the reconstruction of map from the first dataset, resulting two best 3D classes, which in total contain 67 percent of the whole particles.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

2veq

chain_id: A, residue_range: 48-608

1nex

chain_id: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Cross-correlation coefficient
Output model

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

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