ICMT is displayed as a progression from primary sequence alignment, to the effects of scanning mutagenesis (bar graphs), to observed secondary structure (α-helices depicted as ribbons), to the assigned role of amino acids. Results from scanning mutagenesis experiments12 are plotted above the sequence alignment as a bar graph showing the reduction of specific activity in comparison to wild-type ICMT, with 0% representing wild-type activity and 100% reflecting no detectable activity (indicated by a horizontal dashed line). The inset key denotes the colouring of the bar graph according to the functional role of the amino acid inferred from the atomic structure. Labelled brackets above the secondary structure denote the general function of the indicated regions of the primary sequence. In the bar graph: magenta, amino acids that contact AdoHcy; red, amino acids that line the lipid-binding cavity; blue, arginine residues that are proposed to form hydrogen bonds with the carboxylate of the prenylcysteine substrate, and the residues that position them; grey, residues proposed to form hydrogen bonds to the methyl of AdoHcy in the transition state; hydrogen bonds made with backbone atoms are indicated by parentheses surrounding the amino acid label. The mutagenesis data are derived from experiments using A. gambiae ICMT12, and are normalized for expression level. Except where noted, the mutations were alanine substitutions. In some cases, leucine substitutions (L) were made (for example, when the wild-type amino acid was a glycine or alanine). The data represent triplicate measurements for each mutation and the mean s.d. is 11%. Gaps in the bar graph indicate amino acid positions that were not analysed by mutagenesis. Based on size-exclusion chromatography that was performed for each of the mutants, only the E141A mutation was found to be notably destabilizing (asterisk). A few mutations increased the activity relative to wild-type; these are shown as exhibiting 0% reduction in activity. The amino acid sequences included in the alignment are: T. castaneum ICMT (beetle ICMT), human (Hs), A. gambiae (Ag), Saccharomyces cerevisiae (Sc), and Arabidopsis thaliana (At) (UniProt accession numbers: D6WJ77, O60725, Q7PXA7, P32584 and Q93W54, respectively). The alignment is coloured according to the ClustalW convention.