Initial holo structures for ligands coupled to proteins as well as the solo ligands (used for the ligand in water leg of the thermodynamic cycle) were reused from the relative binding free energy study . Initial structures for protein apo simulations were constructed from apo crystal structures (PDB IDs 3o17 (jnk1), 1wfc 53 (p38α), 4htz 55 (pde2), 1h27 33 (cdk2), 1r1w (cmet), 3zsl 41 (galectin)), where available. For these structures missing residues were modeled in and the amino acid protonation states were adjusted to match those of the holo structures. Energy minimization and equilibration with NVT and NPT simulations in the presence of solvent and ions were also carried out (in the same manner as in the core part of the protocol below) to relax the reconstructed residues. Finally, the apo protein structures were extracted from the last frame of the NPT simulations (or, in cases where no residue reconstruction was necessary, from the protonation adjustment stage) and were used to initialize the protein-only systems. As no apo crystal structure was available for tyk2, a holo structure with the ligand removed was used instead.