Requirements on Experimental Structures and Binding Affinity Data for FEP+

When choosing a system for an FEP study, the experimental structures used should meet the following criteria:

• There is at least one high-quality crystal structure of each ligand from the series.
• All residues close to the binding pocket are clearly resolved.
• There are no chain breaks near the binding pocket. (Chain breaks far away from the binding pocket can be built from other crystal structures of the same protein.)
• A reasonable number of ligands with high-quality crystal structures is available: more ligands should yield better results.
• There is a reasonable expectation of a conserved binding mode across the series.
• Interactions between the core part of the ligands and the protein are strong enough to maintain the binding pose in an MD trajectory.
• There are minimal tautomeric, ionization-state, and stereochemical uncertainties across the series.
• High reliability experimental binding data from the same assay is available for all compounds.
• Assay data and crystal structures are for the same protein construct.

Failing to meet these criteria doesn’t necessarily guarantee failure, but it does make it more likely. In general, the cleanness of the experimental data is much more important than the identity of the target or the type of modification.

However, you should avoid the following systems:

• Systems where ligands form different interactions with metals in the protein. Interactions between metals and different chemical groups in the ligands are not well captured by the fixed-charge force field.