2019-12-11RELPDBePDBePDBe2017-12-302018-01-102018-08-012019-12-11Biotechnology and Biological Sciences Research CouncilBB/J007595/1United KingdomCryo-EM structure of the core Centromere Binding Factor 3 complexZhang WJLukoynova NMiah SVaughan CKZhang WLukoynova NMiah SLucas JVaughan CKInsights into Centromere DNA Bending Revealed by the Cryo-EM Structure of the Core Centromere Binding Factor 3 with Ndc10.Cell RepUS24744754201830021170doi:10.1016/j.celrep.2018.06.0682211-1247EMD-4241associated EM volumeCryo-EM structure of the core Centromere Binding Factor 3 complexEMD-0051other EM volume6fe8FULLOVERLAPThe core Centromere Binding Factor 3 complexThe core Centromere Binding Factor 3 complex0123The 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.Saccharomyces cerevisiaeSaccharomyces cerevisiae0.22Centromere DNA-binding protein complex CBF3 subunit BSaccharomyces cerevisiae0.068454125Model is numbered according to2Saccharomyces cerevisiaeLEVOMGGSSHHHHHHSSGLVPRGSHMKLITASSSKEYLPDLLLFWQNYEYWITNIGLYKTKQRDLTRTPANLDTDTEECMFWMN
YLQKDQSFQLMNFAMENLGALYFGSIGDISELYLRVEQYWDRRADKNHSVDGKYWDALIWSVFTMCIYYMPVEKLAEIFS
VYPLHEYLGSNKRLNWEDGMQLVMCQNFARCSLFQLKQCDFMAHPDIRLVQAYLILATTTFPYDEPLLANSLLTQCIHTF
KNFHVDDFRPLLNDDPVESIAKVTLGRIFYRLCGCDYLQSGPRKPIALHTEVSSLLQHAAYLQDLPNVDVYREENSTEVL
YWKIISLDRDLDQYLNKSSKPPLKTLDAIRRELDIFQYKVDSLEEDFRSNNSRFQKFIALFQISTVSWKLFKMYLIYYDT
ADSLLKVIHYSKVIISLIVNNFHAKSEFFNRHPMVMQTITRVVSFISFYQIFVESAAVKQLLVDLTELTANLPTIFGSKL
DKLVYLTERLSKLKLLWDKVQLLDSGDSFYHPVFKILQNDIKIIELKNDEMFSLIKGLGSLVPLNKLRQESLLEEEDENN
TEPSDFRTIVEEFQSEYNISDILSSuppressor of kinetochore protein 1Saccharomyces cerevisiae0.0225584511Saccharomyces cerevisiaeLEVOMGVTSNVVLVSGEGERFTVDKKIAERSLLLKNYLNDMHDSNLQNNSDSESDSDSETNHKSKDNNNGDDDDEDDDEIVMPV
PNVRSSVLQKVIEWAEHHRDSNFPDEDDDDSRKSAPVDSWDREFLKVDQEMLYEIILAANYLNIKPLLDAGCKVVAEMIR
GRSPEEIRRTFNIVNDFTPEEEAAIRRENEWAEDRGSCentromere DNA-binding protein complex CBF3 subunit CSaccharomyces cerevisiae0.0608999611Saccharomyces cerevisiaeLEVOMGPSFNPVRFLELPIDIRKEVYFHLDGNFCGAHPYPIDILYKSNDVELPGKPSYKRSKRSKKLLRYMYPVFATYLNIFEY
SPQLIEKWLEYAFWLRYDCLVLDCFKVNHLYDGTLIDALEWTYLDNELRLAYFNKASMLEVWYTFKEYKKWVIDSVAFDE
LDLLNVSNIQFNIDNLTPQLVDKCLSILEQKDLFATIGEVQFGQDEEVGEEKDVDVSGANSDENSSPSSTIKNKKRSASK
RSHSDNGNVGATHNQLTSISVIRTIRSMESMKSLRKITVRGEKLYELLINFHGFRDNPGKTISYIVKRRINEIRLSRMNQ
ISRTGLADFTRWDNLQKLVLSRVAYIDLNSIVFPKNFKSLTMKRVSKIKWWNIEENILKELKVDKRTFKSLYIKEDDSKF
TKFFNLRHTRIKELDKSEINQITYLRCQAIVWLSFRTLNHIKLQNVSEVFNNIIVPRALFDSKRVEIYRCEKISQVLVIG
SRSGSENLYFQGSKRRWKKNFIAVSAANRFKKISSSGALsingleParticleparticle0.128200.0NaClsodium chloride15.0Tris base2.0DTTQuantifoil R1.2/1.3GOLD300GLOW DISCHARGEETHANE100277FEI VITROBOT MARK IThe sample is homogeneous and well-dispersed on grids.FEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN3002.747170.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENGATAN K2 SUMMIT (4k x 4k)COUNTING112360.44.6GATAN K2 SUMMIT (4k x 4k)COUNTING1-40111010.3754.061The selected images were high-pass filtered and normalized.139303CTFFIND4.0CTF parameters were estimated using CTFFIND4An ovoid generated from SPIDER was used as an initial model for 3D classification.4C1FOURIER SPACE4.1FSC 0.143 CUT-OFFRELION2.03D 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.69392ANGULAR RECONSTITUTIONRELION2.0ANGULAR RECONSTITUTIONRELION2.0417348RELION2.0The automatically picked particles were screened manually followed by reference-free 2D classification, which yielded 69,392 particles for subsequent 3D classification.2The selected images were high-pass filtered and normalized.289281CTFFIND4.0CTF parameters were estimated using CTFFIND4An ovoid generated from SPIDER was used as an initial model for 3D classification.2C1FOURIER SPACE3.7FSC 0.143 CUT-OFFRELION2.0187, 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.187606ANGULAR RECONSTITUTIONRELION2.0ANGULAR RECONSTITUTIONRELION2.0394038RELION2.0After 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.2VEQA48-6081NEXAAB INITIO MODELCross-correlation coefficientREALemd_4241_msk_1.mapemd_4241_half_map_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
200
200200
0
00200200200211.99998211.99998211.9999890.090.090.0XYZ-0.022654750.0553388860.000326001520.0035032581.061.061.06Half map of the final map, which was used for FSC calculation.emd_4241_half_map_2.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
200
200200
0
00200200200211.99998211.99998211.9999890.090.090.0XYZ-0.0184129360.0560055970.000328977770.003497861.061.061.06The other half map of the final map, which was used for FSC calculation.