simulations were performed using the package with the ff99SB force field for proteins and GAFF for ligands. RESP partial charges for the two FK866 supplier ligands FAS and CRB were derived using GAUSSIAN03 at the theory level and the antechamber program. CDK2 and CDK4 were solvated in a cubic solvent box so that the distance between every solute atom and the box boundary was at least neutralised by adding counter ions. Water molecules were treated using the TIP4P-Ew water model, a reparameterization of TIP4P with Ewald summation five buried crystal waters for CDK2 and four crystal waters present in equivalent positions in CDK4 were kept in the simulations. Before the simulations the systems were energy minimized, initially by steepest descent followed by conjugate gradient minimization. Then the energy-minimised complexes were equilibrated by heating followed density equilibration, both with weak restraints for all protein residues and 500 ps constant pressure equilibration. For all simulations the SHAKE algorithm was used to constrain bonds between hydrogens and heavy atoms, Langevin dynamics were used for temperature control. Amber tools were used for the analysis of the MD runs, for example the presence or absence of H-bonds was tested using the ptraj hbond command with default settings. Note that with this TI setup, the total system charge changes during a single transformation step, while overall charge neutrality for the thermodynamic cycle is of course maintained. Charge-change TI calculations involve some additional practical challenges when compared to charge neutral ones, as electrostatic interactions are strong and long-ranged, leading to potential convergence problems. Nevertheless, the PME long-range electrostatics treatment used here allows for simulations of such net-charge changes. CDK4 had escaped structural characterisation by X-ray crystallography for a long time, achieved a major breakthrough and solved its structure in complex with cyclin D1 and cyclin D3, respectively. These experimentally determined CDK4 structures are proposed to represent an intermediate, not fully activated state and none of the as yet published structures contains a small molecule inhibitor in the ATP binding site. Before experimentally determined CDK4 structures became availab
