C3-P-12“Minispindle” as a tool for analysis of individual microtubule behavior in a mitotic spindle

The V-ATPase from Thermus thermophiles is a rotary enzyme, that mediates the coupling between ATP synthesis in the soluble part (V1) and proton translocation across membrane through the membrane-embedded part (Vo). Atomic resolution structural information of the soluble catalytic region has not been obtained yet, and little is known about the high-resolution structure of the Vo. Single particle analysis by cryo-electron microscopy has advanced to a technique for high-resolution structural analysis recently. Thus, now we are trying to get high-resolution structures of both V1 and VoV1 (intact enzyme) using a cryo-electron microscope (Titan Krios) equipped with a direct electron detector (Falcon II). We prepared grids for cryo-electron microscopy using Vitrobot (FEI) with the recombinant V1 and the Vo solubilized by DDM. We used thin carbon-backing for Vo and collected the images from both samples using EPU (FEI). For the efficient data collection by EPU, we used Quantifoil R2/4 made by Mo with 200 mesh. It is known that the particles on the images by cryo-electron microscopy are subjected to a beam-induced movement and so we applied motion collection before the image processing by Relion. We will show the resulted structures and discuss about possible improvements to the technique. Images of the VoV1 holoenzymes C3-P-12 doi:10.1093/jmicro/dfv315