We observed a substantial difference between the conformational flexibility of the uncomplexed cyclodextrins in solution when simulated with GAFF v2.1 versus SMIRNOFF99Frosst and GAFF v1.7. With SMIRNOFF99Frosst and GAFF v1.7, the average RMSD of βCD, relative to the initial structure, is between 2.0-2.5 Å over 43 μs of unrestrained simulation, while with GAFF v2.1, the average RMSD is <1.0 Å (Figure ). Not only are the RMSDs greater for SMIRNOFF99Frosst and GAFF v1.7, but there is greater variance in their RMSDs compared to GAFF v2.1, indicating greater flexibility. This large difference in structural fluctuations is clearly visible in the structure overlays also shown in the figure, which shows that GAFF v2.1 is the only one of the three force fields that leads to maintainance of a clearly defined binding cavity. In this respect, it is similar to the q4md-CD force field , which was designed specifically for cyclodextrins and which also maintains a relatively well-defined cavity . This difference may be further analyzed by considering the "flip" pseudodihedral O2 n -C1 n -C4 n+1 -O3 n+1 , which characterizes the orientation of glucose monomers relative to their neighbors. An angle of 0°c orresponds approximately to a glucose that forms part of a cylindrical wall of the binding cavity, while an angle of ± 90° indicates a glucose that has flipped to put its plane parallel to the top and bottom of the cylinder, partly filling the cavity. This dihedral is tightly distributed in GAFF v2.1, with all seven instances having a Gaussian-like distribution centered around -10° (Figure ). GAFF v1.7 and SMIRNOFF99Frosst display, a mixed population of monomers both aligned with, and perpendicular to, the cyclodextrin cavity. In particular, during a single 1 μs simulation, each monomer will sample conformations at 0° and ±90°, as indicated by the timeseries in Figure