Multiple sequence alignment and secondary structure elements assignment. The alignment of 7 representative T. thermophila P450 genes and one predicted P. tetraurelia P450 gene (CYP693A1, ParameciumDB accession No. GSPATP00036495001) with the mamalian CYP3A4 protein sequence. Substrate recognition sites (SRSs) 1–6 were manually determined. The heme-binding region (FXXGXRXCXG), PERF domain (PXRX) and K-helix (EXXR) were indicated with blue triangles respectively. The alpha helices are marked as alpha or eta based on the automatic assignment according to the template of CYP3A4 protein structure in the program ESPript.