2. Materials and Methods2.1. AMBER(RESP)A single cholesterol structure was model-built and optimized using Gaussian-09 [31] at the AM1 and B3LYP/3-21G* level. The minimized geometry was considered at the HF/6-31G* level and electrostatic potentials (ESPs) were computed for subsequent best RESP assignment of atomic partial charges using ANTECHAMBER together with the GAFF force field (AMBER [18, 32] version 11). A single copy of RESP/GAFF-parameterized cholesterol was loaded into XLEAP and output in appropriate AMBER formats (prmtop/inpcrd), then minimized (2500 steps, cutoff 20?), and heated to 300K target temperature (vacuum conditions) using Langevin dynamics, collision frequency �� = 1ps?1, 12? cutoff, a time step of 1fs, and no constraints on any bonds (i.e., SHAKE switched off).

Identical conditions were applied during 5ns of MD simulation where conformational snapshots were saved every 5000 steps to create a sample of 250 structures. 2.2. AMBER(bcc)Cholesterol was model-built and optimized at the HF/6-31G** level using Gaussian-03 [31]. Atomic partial charges were assigned following the approach of AM1-bond charge correction (bcc) available in ANTECHAMBER of the AMBER package [18, 32] (version 8). The optimized structure was minimized (20 steps, steepest descent) and heated to the target temperature of 300K within 100ps of equilibration MD. Production MD over 5ns used a time step of 1fs, SHAKE constraints on XH bonds, a Berendsen thermostat, vacuum conditions without periodicity, and AMBER version 10. 2.3. CHARMMCholesterol parameters were employed as reported previously [33].

A system containing a single copy of cholesterol was set up and heated to 300K based on straight dynamics and CHARMM36 all-hydrogen lipid topology/CHARMM27 all-hydrogen lipid parameters [19]. Production MD was extended over a period of 5ns using a time step of 1fs, no SHAKE constraints, and the default cutoff of 12? specified in the cholesterol parameter file. 2.4. GROMACSCholesterol parameters were used as reported previously [34]. A single molecule of cholesterol was put into a cubic box (58.219?3) and simulated at constant volume using periodic boundary conditions. All bonds were constrained [35], a time step of 2fs was applied, total translation/rotation was periodically removed every 1000 Carfilzomib steps, neighbour lists were updated every 5 steps, a cutoff of 10? was used, and the system was maintained at 300K target temperature by means of a Nose-Hoover thermostat. 2.5. PCAAll 250 snapshots collected by all the 4 different MD simulations were automatically converted from pdb format to xyz format and analyzed frame by frame for geometrical relationships (bonds/angles/dihedrals) with the help of TINKER [25] (module ANALYZE, option d) using the MM3 force field [36].