2022-01-19RELRCSBRCSBRCSB2021-10-162022-01-192022-01-192022-01-19Howard Hughes Medical Institute (HHMI)United States13pf E254A microtubule from recombinant human tubulin decorated with EB3LaFrance BJGreber BJZhang RMcCollum CNogales ELaFrance BJRoostalu JHenkin GGreber BJZhang RNormanno DMcCollum COSurrey TNogales EStructural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.Proc.Natl.Acad.Sci.USAUS119202234996871doi:10.1073/pnas.21149941191091-64900040PNASA6LaFrance BJRoostalu JHenkin GGreber BJZhang RNormanno DMcCollum CSurrey TNogales EStructural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubulesBioRxivUS2021doi:10.1101/2021.08.13.456308EMD-25159associated EM volumeEMD-25156other EM volumeEMD-25157other EM volumeEMD-25158other EM volumeEMD-25160other EM volumeEMD-25161other EM volume7sj9FULLOVERLAP13pf E254A microtubule decorated with EB313pf E254A microtubule decorated with EB30123Homo sapiens0.102Tubulin alpha-1B chainHomo sapiens0.050975288999999996Trichoplusia niLEVOMRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIHHHHHHGGGDDSFNTFFSETGAGKHVPRAVFVDLEPTV
IDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLDRIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLM
ERLSVDYGKKSKLEFSIYPAPQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLISQI
VSSITASLRFDGALNVDLTAFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMA
CCLLYRGDVVPKDVNAAIATIKTKRSIQFVDWCPTGFKVGINYQPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHK
FDLMYAKRAFVHWYVGEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEYTubulin beta-3 chainHomo sapiens0.0512763669999999966Trichoplusia niLEVOMREIVHIQAGQCGNQIGAKFWEVISDEHGIDPSGNYVGDSDLQLERISVYYNEASSHKYVPRAILVDLEPGTMDSVRSGA
FGHLFRPDNFIFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKECENCDCLQGFQLTHSLGGGTGSGMGTLLISKVREEYP
DRIMNTFSVVPSPKVSDTVVEPYNATLSIHQLVENTDETYCIDNEALYDICFRTLKLATPTYGDLNHLVSATMSGVTTSL
RFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTARGSQQYRALTVPELTQQMFDAKNMMAACDPRHGRYLTVATVFRGR
MSMKEVDEQMLAIQSKNSSYFVEWIPNNVKVAVCDIPPRGLKMSSTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTG
EGMDEMEFTEAESNMNDLVSEYQQYQDATAEEEGEMYEDDEEESEAQGPKENLYFQMicrotubule-associated protein RP/EB family member 3Homo sapiens0.0320211662Escherichia coli BL21LEVOMAVNVYSTSVTSENLSRHDMLAWVNDSLHLNYTKIEQLCSGAAYCQFMDMLFPGCVHLRKVKFQAKLEHEYIHNFKVLQA
AFKKMGVDKIIPVEKLVKGKFQDNFEFIQWFKKFFDANYDGKDYNPLLARQGQDVAPPPNPGDQIFNKSKKLIGTAVPQR
TSPTGPKNMQTSGRLSNVAPPCILRKNPPSARNGGHETDAQILELNQQLVDLKLTVDGLEKERDFYFSKLRDIELICQEH
ESENSPVISGIIGILYATEEGFAPPEDDEIEEHQQEDQDEYGUANOSINE-5'-TRIPHOSPHATE0.000523179999999999912GTPMAGNESIUM ION2.4305e-0512MGhelicalhelicalArray5.56.880 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8C-flat-1.2/1.3COPPER300GLOW DISCHARGEETHANE-PROPANE55 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the gridFEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN3002.60.52.564000.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENGATAN K2 SUMMIT (4k x 4k)SUPER-RESOLUTION40.01Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames181.64-0.19C13.8FSC 0.143 CUT-OFFFREALIGN9.1110008CTFFIND4RELION3.1CTFFind4 and CTFRefine were used within the RELION3.1 framework.Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box.EMD-7973For the 13pf model, EMD-7973 was low pass filtered to 50A to serve as an initial model. In house reconstructions of 12-15pf at 50A resolution were used to separate by PF.NOT APPLICABLEFREALIGN9.116DPURIGID BODY FITCorrelation CoefficientREAL115.0