Amber MD simulation of protein-ligand complex in waterbox 

 Step1 - Preprocees the protein pdb File Check protonation state of the protein using propka3 (It comes by default with python versions>3 in linux) >> propka3 pro.pdb or also can protonate the protein using H++ server To separete the ligands from the downloaded protein pdb file from RCSB database 

>> pdb4amber pro.pdb > pro_amber.pdb

Step2: Preparation of the parameter files: generate the parameters files for small moleclue/ligand/substarte present in your protein 

>> antechamber -i lig.pdb -fi pdb -o lig.prep -fo prep -j 4 -at amber -c bcc -nc 0 

Check with 'antechamber -h' for more options Can use command 'antechamber –i lig.out –fi gout' if the input file is from Gaussian 

>> parmchk2 -i lig.prep -f prepi -o lig.frcmod 

Step3: Set a cubic water box containing protein and small molecule Source to all the force field file that are needed to create the system. You can keep these files in the same folder or make a source to their location. You can run the command below by putting the lines in tleap.in file Then run 'tleap -f tleap.in' 

>> source leaprc.protein.ff19SB 

>> source leaprc.gaff2 

>> source leaprc.water.tip3p 

>> source leaprc.GLYCAM_06EPb 

>> source leaprc.GLYCAM_06j-1 

>> loadamberprep lig.prep 

#.prep and .frcmod files are generated by the Antechamber program 

>> loadamberparams lig.frcmod 

>> mol = loadpdb pro_amber.pdb #load the protein+ligand complex 

>> center mol #set the origin to the center of the box 

>> solvatebox mol TIP3PBOX 10 #Keep the protein 10 Angstrom apart from the water box 

>> charge mol #To know the charge of the system 

>> addions mol Cl- 0 #It will neutralize the system to charge 0 if there are any charges in the system 

>> addions mol Na+ 0 

>> saveamberparm mol pro_n.prmtop pro_n.inpcrd 

>> savePdb mol pro_n.pdb #Save the system containing the water box 

>> charge mol #To know the charge of the system after adding the ions 

>> quit 

Step4: preparation of the input files to run simulation in HPC

Step6: Analysis of the trajectories using cpptraj Can run by putting lines below in the 'analysis.in' and then run like this 'cpptraj analysis.in' Processing of the trajectory file for checking the trajectory files in VMD Load the topology or pdb file first and then the trajectory file (in .dcd or .nc format) on top of it parm pro.prmtop for i=1;i<5;i++; trajin md$i.nc 0 last 100 ##You can write the input in your own style and can be more creative here done autoimage strip :WAT ##Remove water molecules/ions/ligands to decrease the trajectory file size strip :Na+ trajout short_traj_100del.nc onlyframes 1-50000 Calculation of RMSD parm pro.prmtop for i=1;i<5;i++; trajin md$i.nc 0 last 100 done reference Cryst.pdb ##reference file or a pdb file autoimage rmsd @CA reference out rmsd.out @CA Other advanced calculations can be done using pytraj/mdtraj in Jupyter Notebook or by using MDanalysis