The systems were energy minimized initially by steepest descent followed by conjugate gradient minimization. Then the energy-minimised complexes were equilibrated by 50 ps heating from 0 K to 300 K followed by 50 ps density equilibration, both with weak restraints for all protein residues and 500 ps constant pressure equilibration. Production runs were run for 5 ns with 2 fs time steps. 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 Calicheamicin γ1 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 YHO-13351 (free base) 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, but in 2009 Day et al. and Takaki et al. 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 available, CDK4 homology models based on experimentally determined structures of CDK 2 and/or CDK6 were commonly used for computational studies such as ligand docking e.g. and molecular dynamics simulations. Most small molecule CDK4 inhibitors are competitive inhibitors for ATP and target the active form of CDK4. Hence, CDK4 homology models representing the active form still have been used in recent ligand docking studies, despite the availability of experimentally determined CDK4 structures. To take advantage of the new Xray structures we op
