In summary, over the course of the designed trajectory, we observe two jumps in the IN/OUT ratio, with enrichment towards the catalytically preferred "IN" conformation. The first of these is between GNCA4-WT and GNCA4-12 (Figure ). GNCA4-12 is the best variant from our initial FuncLib study. As shown in Figure of ref. , the structural changes between GNCA4-12 and the wild-type enzyme are minimal. However, our analysis of the conformational space of W290 (Figure ) shows enrichment of W290 towards catalytically productive conformations. This in turn likely leads to the stabilization of the "IN" conformation in GNCA4-12, with this stabilization being further enhanced in V4-4 (which also includes the C-terminal polypeptide segment). This segment closes over the active site and restricts its volume contributing to stabilization of the "IN" conformation.