I am trying to calculate binding affinity using MMPBSA calculation for protein-ligand system. I want to include one explicit water molecules in the calculations. For that, I created topology file for ligand, protein+1 water molecule using following lines.

$ cpptraj parm ../../file_preparation/complex_solvated.prmtop trajin sim1.dcd strip :Na+ outprefix strip trajout strip.dcd run exit

        2. After removing the counter ions, I topology file for protein+ligand+(one closest water molecule near ligand)    $ cpptraj parm ./strip.complex_solvated.prmtop trajin strip.dcd closest 1 :LIG outprefix complex_1_wat trajout complex_1_water.mdcrd trajout complex_1_water.pdb run exit

         3. Using below lines I am creating topology file for protein+one water molecule $ cpptraj parm ./complex_1_wat.strip.complex_solvated.prmtop trajin complex_1_water.mdcrd strip :LIG outprefix protein_1_water run exit

          4. I created the topology file for ligand molecule. $ cpptraj parm ./complex_1_wat.strip.complex_solvated.prmtop trajin complex_1_water.mdcrd strip !:LIG outprefix ligand run exit Finally, I am trying to run MMPBSA.py (from amber18) as follows:

MMPBSA.py -O -i mmpbsa.in -o FINAL_RESULTS.dat -cp complex_1_wat.strip.complex_solvated.prmtop -rp protein_1_water.complex_1_wat.strip.complex_solvated.prmtop -lp ligand.complex_1_wat.strip.complex_solvated.prmtop -y complex_1_water.pdb I got the following error: Loading and checking parameter files for compatibility... mmpbsa_py_energy found! Using /usr/local/apps/amber18/bin/mmpbsa_py_energy cpptraj found! Using /usr/local/apps/amber18/bin/cpptraj Preparing trajectories for simulation... Error: Could not initialize action [rmsd] 1 errors encountered reading input. Error: Error(s) occurred during execution.   File "/usr/local/apps/amber18/bin/MMPBSA.py", line 99, in     app.file_setup()   File "/usr/local/apps/amber18/lib/python2.7/site-packages/MMPBSA_mods/main.py", line 156, in file_setup     self.mpi_size, str(external_progs['cpptraj']), self.pre)   File "/usr/local/apps/amber18/lib/python2.7/site-packages/MMPBSA_mods/make_trajs.py", line 134, in make_trajectories     traj.Run(pre + 'normal_traj_cpptraj.out')   File "/usr/local/apps/amber18/lib/python2.7/site-packages/MMPBSA_mods/make_trajs.py", line 687, in Run     raise TrajError('Error running %s' % self.program) AttributeError: 'Trajectory' object has no attribute 'program' Exiting. All files have been retained. Can someone please help me to identify the error.