To further confirm the mode of binding, crystallographic studies were initiated. Soaking of 1 into preformed crystals of MutSβ bound to mismatch DNA (IDL of 2 bases, based on PDB: 3THY) and ADP in the MSH2 pocket resulted in a dataset containing electron density for the ligand in the MSH3 ATP binding pocket. This crystal structure contained two molecules of MutSβ in the asymmetric unit and we observed different ligand conformations in the two individual MSH3 subunits (Figure and Figure ). A crystallographic sulfate ion located at the α-phosphate position was observed in one of these cases. Based on recent structural data, we believe that the modelled sulphate belongs to a loosely bound HEPES molecule, which is fully visible in the electron density when the signature loop is closed (unpublished results). Differences in the ligand conformation of 1 result from variation in the angle of the chlorophenyl group in relation to the pteridine moiety. This is likely a result of different conformations in the adjacent helix containing the Walker-Amotif which also results in slightly different signature loop conformations of MSH2. We observed similar binding to Val878 in our ligand structures to those previously reported for the Adenine motif of ADP bound to MSH3 (PDB: 8OM9). However, in contrast to ADP, rather than occupying the phosphate binding region we observed a binding interaction between the piperazine and Asp875. This interaction was further verified with an in silico analysis of 1 using a fragment molecular orbital (FMO) method. A minimization of 1 was performed followed by fragmentation and MP2 QM calculations. Based on the results of this analysis, the strongest interaction between 1 and MSH3 was determined to be between the piperazine and the Asp875, strengthening the validity of the hypothesized conformation of the ligand (Figure ).