2021-05-19RELRCSBRCSBRCSB2021-03-172021-05-122021-05-122021-05-19Howard Hughes Medical Institute (HHMI)David AgardUnited StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM031627United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118099United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01 GM105537United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103533United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM083960United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM109824United StatesNational Science Foundation (NSF, United States)1144247United StatesNational Institutes of Health/Office of the Director1S10OD020054United StatesNational Institutes of Health/Office of the Director1S10OD021741United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM124149United StatesNational Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124169United StatesDepartment of Energy (DOE, United States)DE-AC02-05CH11231United StatesSingle-particle reconstruction of a dimer of the Yeast gamma-TuSCBrilot AFLyon ASZelter AViswanath SMaxwell AMacCoss MJMuller EGSali ADavis TNAgard DABrilot AFLyon ASZelter AViswanath SMaxwell AMacCoss MJMuller EGSali ADavis TNAgard DACM1-driven assembly and activation of yeast gamma-tubulin small complex underlies microtubule nucleation.ElifeUS10202133949948doi:10.7554/eLife.651682050-084XEMD-23638other EM volumeEMD-23637other EM volumeEMD-23636other EM volumeEMD-23635other EM volumeEMD-23639associated EM volumeSingle-particle reconstruction of a dimer of the Yeast gamma-TuSCSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC0123Saccharomyces cerevisiaeSpindle Pole BodySpodoptera frugiperdaSF90.6Tub4p (gamma-tubulin)Saccharomyces cerevisiaeSpodoptera frugiperdaLEVOMGGEIITLQAGQCGNHVGKFLWSQLAKEHAIGTDGLSQLPDSSTERDDDTKPFFRENSRNKFTPRAIMMDSEPSVIADVENTFRGFFDPRNTWVASDGASAGNSWANGYDIGTRNQDDILNKIDKEIDSTDNFEGFQLLHSVAGGTGSGLGSNLLEALCDRYPKKILTTYSVFPARSSEVVVQSYNTILALRRLIEDSDATVVFDNASLLNISGKVFRNPNIDLQHTNQLISTIISSVTNSIRFPSYMYSSMSSIYSTLIPSPELHFLSPSFTPFTSDYIHDDIAHKGHSSYDVMLDLLDPSNSLVSTAMNNPTYFNVYNTIIGNVEPRQISRAMTKLQQRIKFPSWSSSAMHVNIGRRSPYLPLQPNENEVSGMMLSNMSTVVNVFENACNTFDKVFAKGAFLNNYNVGDLFQSMQNVQDEFAESREVVQSLMEDYVAAEQDSYLDDVLVDDENMVGELEEDLDADGDHKLVSpc97pSaccharomyces cerevisiaeSpodoptera frugiperdaLEVOMEIKEVDDRAELLRYTNNIPLLGKLVNHQPLWSTNPKLKSFSLEKISAPDQRRVQEALVVKDLLNVLIGLEGTYIRYFNDYEPSDPETPIEFKIAKKMDPSFKTFSRRIVRYGKQYMILTRAYEKWSDTSFGMVLQRFAYEIRRFLEDVYLKTLVERLERDFNKVPNFSIRELEQIINETEVNKQMELLYNIYEEIFREIEERRTNQSSQEDFNNFMDSMKNESSLHLRLMVAFDTTVYPVPKGGAILKIFQQKILENLGDRSSVMFLKKLLNNISQDYCTMLYEWLTQGILNDPYQEFMTYDDLEGKTDNIFDTRDRAWDTQYFIRKDVLLRDCDSEEDKNLLFKMLRTGILLKVVRASLQIPTIPSNSSDITIQEINDFADLMEGSNLELYVDKCYSRANEIFLKLFFQGYDLINVLKHLQQIFLGYQSGHNVLKFLTKNMGELTKHYRNDNNANYDKLLQNFELERQSENPNNLMRQLLMIQFDTETLPQVLSHYLQIYPEVPENNSANDDSDPLMHANNFKNMNAILFDELSKERTGAYHGSNLELYTPKSAIYHLKFDINIPYPLNIIISRTCMIKYQIILRYQLVLQYHSRLLDETWMDLNKTPSWKYRGYSHTVKRRIVRATRVLHAKMNHFIKTIMEYFNQNVIDKEVYSLEKCYRNPTLAVAIQNELEGGLTNIMTNRCLSDLIPLQLQIFDIVYKFCKFIKSMRAKLCQLDPVLYEKHKSGMMKTLNEGYRTNNGGQEDVGYQEDAALELIQKLIEYISNASSIFRKCLINFTQELSTEKFDFYDSSSVDAAGIERVLYSIVPPRSASASSQRSpc98pSaccharomyces cerevisiaeSpodoptera frugiperdaLEVOMELEPTLFGIIEALAPQLLSQSHLQTFVSDVVNLLRSSTKSATQLGPLIDFYKLQSLDSPETTIMWHKIEKFLDALFGIQNTDDMVKYLSVFQSLLPSNYRAKIVQKSSGLNMENLANHEHLLSPVRAPSIYTEASFENMDRFSERRSMVSSPNRYVPSSTYSSVTLRQLSNPYYVNTIPEEDILKYVSYTLLATTSALFPFDHEQIQIPSKIPNFESGLLHLIFEAGLLYQSLGYKVEKFRMLNISPMKKALIIEISEELQNYTAFVNNLVSSGTVVSLKSLYREIYENIIRLRIYCRFTEHLEELSGDTFLIELNIFKSHGDLTIRKIATNLFNSMISLYYEYLMNWLTKGLLRATYGEFFIAENTDTNGTDDDFIYHIPIEFNQERVPAFIPKELAYKIFMIGKSYIFLEKYCKEVQWTNEFSKKYHVLYQSNSYRGISTNFFEIINDQYSEIVNHTNQILNQKFHYRDVVFALKNILLMGKSDFMDALIEKANDILATPSDSLPNYKLTRVLQEAVQLSSLRHLMNSPRNSSVINGLDARVLDLGHGSVGWDVFTLDYILYPPLSLVLNVNRPFGRKEYLRIFNFLWRFKKNNYFYQKEMLKSNDIIRSFKKIRGYNPLIRDIINKLSRISILRTQFQQFNSKMESYYLNCIIEENFKEMTRKLQRTENKSQNQFDLIRLNNGTIELNGILTPKAEVLTKSSSSKPQKHAIEKTLNIDELESVHNTFLTNILSHKLFATNTSEISVGDYSGQPYPTSLVLLLNSVYEFVKVYCNLNDIGYEIFIKMNLNDHEASNGLLGKFNTNLKEIVSQYKNFKDRLYIFRADLKNDGDEELFLLSKSLRsingleParticleparticle0.37.540.0HEPES-KOH100.0Potassium Chloride2.0Magnesium Chloride1.0EGTA1.0GDP2.5GlycerolQuantifoil R1.2/1.3COPPERCARBONHOLEY ARRAYGLOW DISCHARGEETHANE100293FEI VITROBOT MARK IVWhatman #1 Filter papers used.. Sample was a mixture of monomers and dimers.FEI POLARA 300FLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN300100.02.01.03.00.45.031000.039891.0OTHERNITROGENGATAN K2 SUMMIT (4k x 4k)SUPER-RESOLUTION2.53-10020.076.013210917cisTEM pickingcisTEM1.0 betaFinal reconstruction in cisTEMParticle coordinates were semi-automatically picked from filtered and binned images using the e2boxer swarm tool (Tang et al., 2007). Particles were extracted using Relion (Scheres, 2012) with a box size of 384 physical pixels resampled to 96 pixels for initial processing. A dataset of ~50,000 particles from 217 micrographs was used to generate 300 2D classes using Relion 1.3. 23 classes were selected and used in the generation of a gamma-TuSC monomer initial model using the e2initialmodel.py function in EMAN2.1C1FOURIER SPACE4.45FSC 0.143 CUT-OFFcisTEM1.0 Beta506014PROJECTION MATCHINGcisTEM1.0 betaPROJECTION MATCHINGcisTEM1.0 BetaProjection matching in cisTEM2593646.0cisTEM1.0 Beta207.0emd_23639_half_map_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
600
600600
0
00600600600376.02376.02376.0290.090.090.0XYZ-4.2374146.140844-0.00882859250.33785330.62670.62670.6267Single-particle reconstruction of a dimer of the Yeast gamma-TuSC unsharpened unmasked main map.emd_23639_half_map_2.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
600
600600
0
00600600600376.02376.02376.0290.090.090.0XYZ-4.4660785.9869776-0.0094723690.34033460.62670.62670.6267Single-particle reconstruction of a dimer of the Yeast gamma-TuSC half map 1