A closer look at the sequence comparisons in relation to three dimensional structures showed that variation was concentrated in the loops of the ancestors (Supplementary Fig. ). Greater flexibility in the protein scaffold may explain the reduced thermostability of the ancestors relative to P450-TT . Often proteins from thermophiles are shorter than those from mesophiles, by reduction in the length of loops and linkers; however, no such difference was apparent here between P450-TT and the ancestral CYP116B forms. A general comparison of amino acid composition (Supplementary Table ) showed a high degree of similarity between ancestors and extant forms, with a slight shift towards more hydrophobic amino acids in the ancestors compared to P450-TT, as noted previously for the CYP3 family reconstruction .