pf18:WT ratios for select flagellar proteins, abundances for known CA proteins, and criteria used to screen for candidate novel CA proteins. (A) pf18:WT ratios for axonemal and IFT proteins (in this example, the ratios are based on IBAQ scores from replicate 1; see Table S2). Ratios for subunits such as LC7 and LC8, which are present in more than one axonemal structure, are repeated for each structure with which they are associated. The four CA proteins with the highest ratios (calmodulin, enolase, HSP70, and PP1c, in order of decreasing ratios) are located both in the CA and elsewhere in the flagellum. NAP, an unconventional actin previously shown to be a subunit of some inner arm dyneins in the absence of conventional actin, is grouped separately. Our first criterion for considering an uncharacterized protein as a potentially novel CA component was that it had to have a pf18:WT ratio ≤0.2 (red line), ensuring that it is reduced in pf18 axonemes at least as much as PP1c and the known CA-specific proteins. (B) IBAQ values, representing protein abundance, for known CA proteins (values from replicate 1 used as example here). Based on both IBAQ and Top3 values, the least abundant of the known CA proteins in replicate 1 was FAP297. Therefore, our second criterion for considering an uncharacterized protein as a potentially novel CA component was that it had to have an abundance based on IBAQ or Top3 score ≥0.8 of the FAP297 value (red line).