Cryo-ET of Chlamydomonas axonemes

Flagella were isolated from WT Chlamydomonas, the mot7 mutant, and the rescue strain expressing MOT7-BCCP-3HA using the dibucaine method (48). Flagella from the rescue strain were labeled with streptavidin (53). The samples were plunge-frozen as described previously (54) with slight modifications. We used an R3.5/1 holey grid (Quantifoil, Thuringia, Germany) and Cryoplunge-3 (Gatan, USA). We screened cryo-grids with axonemes in proper appearance using a JEM2200FS TEM (JEOL, Japan) [as described in (54)] and acquired the final data using a Titan Krios TEM (Thermo Fisher Scientific, USA) operated at 300-kV accelerating voltage and equipped with a GIF Quantum energy filter and a K2 direct electron detector. Tomographic acquisition of micrographs was carried out at a nominal magnification of 30,000, corresponding to 4.2 Å per pixel, using the SerialEM program (55). Tomograms were analyzed using the IMOD package (56), and then subtomograms were extracted with IMOD and analyzed using our in-house programs using pseudo-ninefold symmetry and 96-nm periodicity as described previously (54). Averaged subtomograms were visualized using IMOD and UCSF Chimera (57).