New simulated annealing approach considering helix bending applied to determine the 8.8 Å structure of 15-protofilament microtubules

X‐ray and Cryo‐ EM structures reveal mutual conformational changes of Kinesin and GTP ‐state microtubules upon binding

“…However, the structural mechanisms explaining how KIF5C distinguishes GTP‐MT from GDP‐MT and the structural features that correspond to faster ATPase cycling are unknown. Therefore, we solved the cryo‐EM structure of KIF5C complexed with GMPCPP‐MT using the previously described real‐space helical reconstruction (Fig A and Supplementary Fig S2A) (Yajima et al , ; Ogura et al , ). We also confirmed the previously solved cryo‐EM structure of the KIF5C–GDP‐taxol‐MT complex to validate the cryo‐EM structures solved here (Fig B, Supplementary Fig S2B and C) (Sindelar & Downing, ; Atherton et al , ).…”

“…Some of the possible reasons for this might be the use of stabilizing reagents for MT, the effect of the bound nucleotide, or the technical limitation to solve the structure of the MT. In this study, we solved the GMPCPP‐bound MT without any other MT‐stabilizing reagent by the single‐particle‐based program (Ogura et al , ). Although clear evidence is shown here to support a different conformation of αβ‐tubulin between MT with and without bound KIF5C, determining the nature of that new conformation requires higher resolution.…”

“…Selected films were digitized with a charge‐coupled device film scanner (Scitex Leafscan45; Leaf Systems) to obtain a final pixel size in the digitized images of 2.5 Å. Image analysis was carried out as previously described (Ogura & Sato, ; Yajima et al , ; Ogura et al , ), with a slight modification in that the high‐pass‐filtered image was cropped into square pieces of 64 × 64 nm instead of 60 × 60 nm to ensure that the size was sufficient to visualize the kinesin–MT complex. For KIF5(Ø)–CPP‐MT and KIF5(Ø)–taxol‐MT, >302,000 and >271,000 tubulin dimers were averaged in total, respectively, to achieve the claimed resolution.…”