2.1.2. Energy Minimization and Heating

The NAMD program [74] v. 2.12 (http://www.ks.uiuc.edu/Research/namd/) was used to minimize the potential energy for 1000 steps using the conjugate gradient method [75,76] then heat the system to the target temperature of 310 K by reassigning velocities from a random distribution at the target temperature every 1000 steps for 20,000 steps with a timestep of 0.002 ps (40 ps). During heating, harmonic positional restraints were placed on non-hydrogen atoms of the solute and constraints [77–79] were applied to maintain equilibrium values for TIP3P geometries and for bond lengths involving hydrogen atoms. The Lennard–Jones (L-J) [80] and electrostatic potential energies had cutoff distances of 10 Å. An energy switching function [81] was applied to L-J interactions between 8 and 10 Å and an isotropic pressure correction accounted for contributions from L-J interactions beyond the cutoff [82]. The particle mesh Ewald (PME) method [83] with fourth order B-spline interpolation for a cubic unit cell and fast Fourier transform (FFT) grid spacing of 1.0 Å along each axis was used to account for electrostatic interactions beyond the cutoff. Consistent with the CHARMM additive force fields for proteins, nucleic acids, lipids, and small molecules [84], carbohydrate 1-4 non-bonded interactions were not scaled (i.e., scaling factor = 1.0). Heating was done under constant pressure with pressure regulated at 1 atm by a Langevin Piston barostat [85]. A 500,000-step (1-ns) unbiased constant particle number/constant pressure/constant temperature (NPT) MD run, followed with a temperature of 310 K maintained by a Langevin thermostat [86] and without positional restraints. The average periodic cell parameters from the last half of this NPT ensemble trajectory (123.7 Å) were used as cell basis vectors for the quadruplicate canonical (NVT) ensemble MD simulations detailed below. These were preceded by minimization and heating as detailed above, with the exception of constant volume (i.e., no Langevin piston barostat) with a box edge length of 123.7 Å.
