Free energy calculations in the HSP90 system. In the HSP90 system, in both EQ and NEQ calculations, independent replicates did not converge to the same free energy estimate within a standard deviation of 0.2 kcal/mol -even with ∼1 µs sampling time per replicate -indicating inadequate sampling (Figure ). In addition to the high standard deviation among replicates and convergence of the cumulative ∆G estimate, several additional factors suggest the presence of one or more slow degrees of freedom in the system.