Unexpectedly, 3 products were resolved in an alternative orientation, termed the "diving" pose, relative to the original fragment F709 (Fig. ) as shown in Fig. , demonstrating the effect of modifying the piperazine ring in Iter 3.5 (Fig. ). These poses rotated by about 90° around the piperazine core (with respect with the original pose) and so that their 5-membered ring displaces waters 2 to 4 while the nearby amide displaces water 1 of the network (Fig. ). There, the 5-membered rings seem to bind mostly via hydrophobic interactions with the amino acids comprising the water cavity. In addition, the cis conformation of the two amides was observed in the divers, while the lateral products displayed a trans conformation. A relatively large protein conformational change is paired with this novel diving binding pose. The ZA-loop adopts a generally more relaxed conformation, resulting in a more voluminous binding site that accommodates the flipped products, thus illustrating that some level of conformational motion is allowed by our crystal system (Fig. ). A striking feature of the diving products is that they all have methyl-substituted piperazine ring. When looking at their closest lateral neighbours based on chemical similarity distance, it seems that this alkyl substitution alone is required to change binding orientation (Fig. ). The relatively low incidence of diving relative to lateral binders also indicates that a high number of experiments are needed to resolve this unexpected pose. The synthesis of the 3 diving binder molecules made use of racemic building blocks (Fig. ), resulting in CRMs containing a mixture of enantiomers and other by-products in unknown ratios. The quality control protocol, however, identified that products with the expected mass were present in the mixture. PanDDA event maps were used instead of traditional electron density maps to fit the compounds. The position of the methyl group around the piperazine ring was ambiguous and required further inspection (Fig. ). Each compound has two stereoisomers with four possible configurations, two on each side of the piperazine. These maps showed a consistent protrusion at the same location, suggesting the presence of an additional group (Fig. ). Other possible locations do not have a similar protrusion and positioning the group there would result in a clash with the protein and/or the compound itself. At the identified position, the methyl group interact with the binding site through hydrophobic interactions (Fig. ).