All single bonds in a molecule that extend from a ring atom are broken in the DeepFMPO process, creating the molecular fragments. The attachment atoms (previously connected with a single bond) are labeled in this step. To calculate ESP similarities the fragments must be aligned in 3D. Here, a conformational search is conducted generating an ensemble of low-energy conformers for all fragments containing rotatable bonds, using the ETKDG method as implemented in RDKit. As default a maximum of ten conformations of each fragment is generated. An anchor group is connected to the fragments' attachment atom and serves as a template in the alignment procedure. The coordinates of the anchor group are fixed in 3D space. The rationale for this step is that ligands containing related fragments typically bind in a similar orientation, and these fragments will frequently make similar ligand-protein interactions. Consequently, to ensure that the fragments are aligned as accurately as possible, an anchor group is attached to the fragments and used in the molecular alignment step. The anchor group was arbitrarily chosen to be a hexazine ring with a methylene linker subunit. This group is of reasonable size for a template and highly unique (i.e., hexazines are never present in drug-like molecules) for easy identification and removal downstream in the process. A few experiments were conducted with other types of structural fragments as anchors to gauge possible conformational effects (vide infra). For each pair of fragments, the pair of conformations with the best shape overlay in terms of the highest shape Tanimoto value are stored. The anchor is then replaced with a hydrogen (see Figure ). In cases where fragments include several labeled atoms, these are replaced with a methyl group. In this manner, all labelled atoms are replaced by a methyl which may be considered neutral in terms of electrostatic similarities. Finally, the ESP Tanimoto value is calculated between the pair of conformers with the best shape alignment (see the "Electrostatic Similarity Calculations" section below).