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(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)

# Analysis of water thermodynamics using Grid Inhomogeneous Solvation Theory of Factor Xa active site. SECTION 2

By Romelia Salomon, Crystal Nguyen, Steven Ramsey, Jonathan D. Gough, Ross Walker, & Tom Kurtzman

### 2) Running the MD calculations to collect frames for GIST

Now that we have relaxed our structure, md_eq-nvt.rst7, we need to run a production MD simulation to sample solvent behavior in the Factor Xa binding site.

For this tutorial, we will run a 30 ns MD simulation in NVT conditions sampling every 1 ps (producing a trajectory with 30,000 frames). Due to space limitations, we have not included the full trajectory and only present the results of the GIST analysis in the following section. It is recommended when applying GIST to collect between 30,000 and 100,000 frames of data (30-100 ns simulation) to ensure converged thermodynamic quantities. For more information on GIST convergence please see: GIST AmberTools

 md-nvt-30ns.in ```Production run 30ns nvt &cntrl imin=0, ntpr = 500, ntwx = 500, ntwr = 500, iwrap=1, ioutfm = 1, ntx=5, irest=1, ntf = 2, ntb = 1, ntc = 2, cut = 9.0, nstlim = 15000000, dt = 0.002, nscm = 1000, temp0 = 300.0, ntt = 3, gamma_ln = 2.0, ntr=1, restraint_wt=100, restraintmask='!:WAT & !@H', ig=-1, &end ```

We can now run this production simulation using pmemd. For example, to run using gpu compiled pmemd (this example requires AMBER to be compiled with gpu support, users could also make use of pmemd.MPI in place of pmemd.cuda, but we do not recommend running the production simulation with default pmemd i.e. 1 cpu since it will take a long time):

\$AMBERHOME/bin/pmemd.cuda -O -i md-nvt-30ns.in -o md-nvt-30ns.out -p fxa.prmtop -c md_eq-nvt.rst7 -r final-prod.rst7 -x prod_30ns.nc -ref md_eq-nvt.rst7

The produced trajectory file will be large, about 21GB, so we have not included it here. We suggest that you run this production simulation to produce the trajectory file. As stated before, this will generate a statistically equivalent trajectory, although not an exactly identical trajectory due to differing random seeds. Our 30ns trajectory, on a GPU GeForce GTX 980, took ~9 hrs

IMPORTANT: Note that an MD trajectory for GIST analysis should be performed with the solute (e.g. the protein) restrained, as shown in the previous input file. GIST mapping is performed on a gridded region defined using x y z coordinates, rather than a relative location (relative to the protein for example), therefore if the solute is translating and rotating the resulting GIST map will provide unwanted results.

Now we have generated our production simulation that we will perform GIST on in Section 3 we will demonstrate how to utilize GIST in cpptraj to produce a solvation thermodynamic map around the active site of Factor Xa.

(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)