DLM is a member of the Scientific Advisory Board of OpenEye Scientific Software, and DLM is an Open Science Fellow with Silicon Therapeutics. CB8-G4 has 3 additional microspecies, a neutral variant (blue), a zwitterionic like form (green), and a negatively charged variant (red) with one of its hydroxyls deprotonated. These microspecies are populated 1.65%, 0.39%, and 0.00% respectively. Table . Summary of binding free energy reference calculations with Parsley 1.2.0 force field for the SAMPL8 host-guest challenge using the Attach-Pull-Release (APR) Method. Table summarizes the calculated free energy and the standard error of the mean (SEM) for each phase (attach, pull, and release) of the APR method, the reference state free energy, and binding free energy (computed as the sum of all phases plus the reference state). In the System ID section, host-guest systems where multiple protonation states were considered include an additional letter P, N, D, or Z. A "P" denotes the guest with a protonated nitrogen with a +1 formal charge, a "N" denotes a guest with a neutral nitrogen, a "D" denotes a guest with a deprotonated hydroxyl oxygen with a -1 formal charge, and a "Z" denotes a guest as a zwitterion. TEETOA-G1-OUT and TEETOA-G1-IN represent retrospective calculations with harmonic restraints on the ethyl groups to orient them away (OUT) or towards (IN) the TEETOA cavity. For some systems, reference calculations were not conducted and are labeled with NM. All energies are reported in units of kcal/mol.