We simulated the RBFE thermodynamic cycle for ligand transformation using a hybrid topology approach for the ligands. We prepared the structures of the individual proteinligand complexes using the same procedure as that used for our ABFE calculations described above. The PDB structures of the complexes for the five ligand pairs with their target proteins are known experimentally, with the exception of the complex for ligand C3d for the Scytalone Dehydratase system (Table ). We generated the structure of this unknown Scytalone Dehydratase complex by aligning the common atoms of the ligand C3d with those of a similar ligand in a known Scytalone Dehydratase complex structure (PDB ID: 5STD ).