View source for 2021 AMBER tutorial 3 with PDBID 1S19

In this tutorial, we will be modeling ligand binding to our receptor using AMBER 16, a molecular dynamics simulation software package created in part by our very own Carlos Simmerling. 

= Initial Structures =

We will be saving all of our initial structures in a directory called '''01.structure'''.

== Protein ==

For the initial protein structure, we will be using the PDB we downloaded from the Protein Data Bank Website (see [https://www.rcsb.org/structure/1s19 here]). Load this PDB into chimera and delete the ligand and any nonstandard residues (ex. waters). Save this as a PDB file, '''1s19_fresh.pdb''', and transfer it to your 01.structure folder.

== Ligand ==

For the ligand, we will again load the 1s19 pdb file from the Protein Data Bank in Chimera. We will delete everything except for the ligand. For this example, the ligand is under the name MC9. To delete everything:

 Select -> Residue -> MC9
 Select -> Invert (all models)
 Actions -> Atoms/Bonds -> Delete

With the ligand isolated, we will add hydrogens and charge. To do this:

 Tools -> Structure Editing -> Add H
 Tools -> Structure Editing -> Add Charge -> (have Amber ff14SB and AM1-BCC selected) -> Ok

Save this as a mol2 file in Chimera and save under the name '''1s19_ligand_dockprep.mol2'''.

'''NOTE'''
It is VERY important to make sure that Chimera adds hydrogens correctly. For this particular ligand the net charge was zero and Chimera was able to model the hydrogens correctly. Many times however, Chimera will add extra hydrogens. If it does, just select the extra hydrogen and go to:

 Actions -> Atoms/Bonds -> Delete

Your ligand will then be protonated and charged correctly.

= Generating Parameters for the Simulation =

Create a new directory called '''02.parameters'''

In order to utilize  Amber for molecular dynamics, parameters for the bio molecules will be needed. Luckily, there have been years of parameter development so parameters for the protein do not have to worried about. However, the small ligand does not have parameters in the standard protein force field. Consequently, we will need to generate a fcmod file specific for the ligand.

To do this, we are going to run the following command:

 antechamber -i ./../01.structure/1s19_ligand_dockprep.mol2 -fi mol2 -o1s19_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0

Gaff2 stands for General Amber Force Field 2, which allows us to generate the parameters for the ligand. The flag at the end '''-nc''' stands for net charge. In our case the net charge is zero so we put a zero there, but change it accordingly for your ligand.

Next, we are going to check these parameters and generate a frcmod file with the following command:

 parmchk2 -i 1s19_ligand_antechamber.mol2 -f mol2 -o 1s19_ligand.am1bcc.frcmod

This command will generate our 1s19_ligand.am1bcc.frcmod file. It is important to keep checking your output files to ensure that everything looks okay. Once checked, we can move onto the next step.

= Building the System with TLeap =

Create a new directory called '''03.leap''' and move into it. 

Up until now, we have separate models for our protein and our ligand. In order to simulate them as a single system, we have to run tleap. TLeap will generate parameter (parm7) and restart (coordinate - rst7) files. To do this, create the file '''leap.in''' and copy in the following script.

 #!/usr/bin/sh 
 
 ###load protein force field
 source leaprc.protein.ff14SB
 ###load GAFF force field (for our ligand)
 source leaprc.gaff
 ###load TIP3P (water) force field
 source leaprc.water.tip3p
 ###load ions frcmod for the tip3p model
 loadamberparams frcmod.ionsjc_tip3p
 ###needed so we can use igb=8 model
 set default PBradii mbondi3
 
 ###load protein pdb file
 rec=loadpdb ./../001.structure/1s19_fresh.pdb
 
 ###load ligand frcmod/mol2
 loadamberparams ./../002.1.parameters/1s19_ligand.am1bcc.frcmod
 lig=loadmol2 ./../002.1.parameters/1s19_ligand_antechamber.mol2
 
 ###create gase-phase complex
 gascomplex= combine {rec lig}
 
 ###write gas-phase pdb
 savepdb gascomplex 1s19.gas.complex.pdb
 
 ###write gase-phase toplogy and coord files for MMGBSA calc
 saveamberparm gascomplex 1s19.complex.parm7 1s19.gas.complex.rst7
 saveamberparm rec 1s19.gas.receptor.parm7 1s19.gas.receptor.rst7
 saveamberparm lig 1s19.gas.ligand.parm7 1s19.gas.ligand.rst7
 
 ###create solvated complex (albeit redundant)
 solvcomplex= combine {rec lig}
 
 ###solvate the system
 solvateoct solvcomplex TIP3PBOX 12.0
 
 ###Neutralize system
 addions solvcomplex Cl- 0
 addions solvcomplex Na+ 0
 
 #write solvated pdb file
 savepdb solvcomplex 1s19.wet.complex.pdb
 
 ###check the system
 charge solvcomplex
 check solvcomplex
 
 ###write solvated toplogy and coordinate file
 saveamberparm solvcomplex 1s19.wet.complex.parm7 1s19.wet.complex.rst7
 quit

To run this script, type:

 tleap -f leap.in

The first section of the script loads the ff14SB, GAFF and TIP3P force fields. In the second part of the script we load our protein, ligand and ligand parameters. The last part of the script creates our parameter and restart files. Most important to us will be the wet complex files. After the files are generated it is very important to check them in Chimera to make sure they look okay. To load parm7 and rst7 files, you have to go to:
 
 Tools -> MD/Ensemble Analysis -> MD Movie --> Choose your parameter and coordinate files to load in

You should get an image that looks like the one below.

[[File:wet_complex.png|thumb|center|400px| Figure 1. Wet complex generated by tleap]]

= Equilibration =

Create a new directory called '''04.equil''' and move into there.

Before we can do a simulation of our system, we first have to do minimizations and equilibrations of it first. We have to do this becuase there could be unfavorable bond angles, bonds or steric clashes that need to be resolved. During this process, we will relax the structure by changing restraints, temperature, pressure, etc. 

== Input Files ==

There will be '''NINE''' steps to the equilibration and all of the input files are copied below. Copy the files into the neme provided immediately above each of the input scripts.

'''01.min.mdin'''
 
 Minimize all the hydrogens
 &cntrl
 imin=1,           ! Minimize the initial structure
 ntmin=2,         ! Use steepest descent Ryota Added
 maxcyc=5000,    ! Maximum number of cycles for minimization
 ntb=1,            ! Constant volume
 ntp=0,            ! No pressure scaling
 ntf=1,            ! Complete force evaluation
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=5.0, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 /


'''02.equil.mdin'''

 MD simulation
 &cntrl
 imin=0,           ! Perform MD
 nstlim=50000      ! Number of MD steps
 ntb=2,            ! Constant Pressure
 ntc=1,            ! No SHAKE on bonds between hydrogens
 dt=0.001,         ! Timestep (ps)
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=1,            ! Complete force evaluation
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask=":!@H=", ! atoms to be restrained
 restraint_wt=5.0, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /


'''03.min.mdin'''

 Minimize all the hydrogens
 &cntrl
 imin=1,           ! Minimize the initial structure
 maxcyc=1000,    ! Maximum number of cycles for minimization
 ntb=1,            ! Constant volume
 ntp=0,            ! No pressure scaling
 ntf=1,            ! Complete force evaluation
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=2.0, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 /


'''04.min.mdin'''

 Minimize all the hydrogens
 &cntrl
 imin=1,           ! Minimize the initial structure
 maxcyc=1000,    ! Maximum number of cycles for minimization
 ntb=1,            ! Constant volume 
 ntp=0,            ! No pressure scaling
 ntf=1,            ! Complete force evaluation
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=0.1, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 /


'''05.min.mdin'''

 Minimize all the hydrogens
 &cntrl
 imin=1,           ! Minimize the initial structure
 maxcyc=1000,    ! Maximum number of cycles for minimization
 ntb=1,            ! Constant volume
 ntp=0,            ! No pressure scaling
 ntf=1,            ! Complete force evaluation
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=0.05, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format 
 /


'''06.equil.mdin'''

 MD simulation
 &cntrl
 imin=0,           ! Perform MD
 nstlim=50000      ! Number of MD steps
 ntb=2,            ! Constant Pressure
 ntc=1,            ! No SHAKE on bonds between hydrogens
 dt=0.001,         ! Timestep (ps)
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=1,            ! Complete force evaluation
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=1.0, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /


'''07.equil.mdin'''

 MD simulation
 &cntrl
 imin=0,           ! Perform MD
 nstlim=50000      ! Number of MD steps
 ntx=5,            ! Positions and velocities read formatted
 irest=1,          ! Restart calculation
 ntc=1,            ! No SHAKE on for bonds with hydrogen
 dt=0.001,         ! Timestep (ps)
 ntb=2,            ! Constant Pressure
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=1,            ! Complete force evaluation
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask="!@H=", ! atoms to be restrained
 restraint_wt=0.5, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /


'''08.equil.mdin''' *Make sure to change the value at restraintmask to the number of residues in your protein including your ligand*

 MD simulation
 &cntrl
 imin=0,           ! Perform MD
 nstlim=50000      ! Number of MD steps
 ntx=5,            ! Positions and velocities read formatted
 irest=1,          ! Restart calculation
 ntc=1,            ! No SHAKE on for bonds with hydrogen
 dt=0.001,         ! Timestep (ps)
 ntb=2,            ! Constant Pressure
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=1,            ! Complete force evaluation
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask=":1-433@CA,C,N", ! atoms to be restrained
 restraint_wt=0.1, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /


'''09.equil.mdin''' *Make sure to change the value at restraintmask to the number of residues in your protein including your ligand*

 MD simulation
 &cntrl
 imin=0,           ! Perform MD
 nstlim=50000      ! Number of MD steps
 ntx=5,            ! Positions and velocities read formatted
 irest=1,          ! Restart calculation
 ntc=1,            ! No SHAKE on for bonds with hydrogen
 dt=0.001,         ! Timestep (ps)
 ntb=2,            ! Constant Pressure
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=1,            ! Complete force evaluation
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 1000,       ! Write to trajectory file every ntwx steps
 ntpr= 1000,       ! Print to mdout every ntpr steps
 ntwr= 1000,       ! Write a restart file every ntwr steps
 cut=  8.0,        ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask=":1-433@CA,C,N", ! atoms to be restrained
 restraint_wt=0.1, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /

== Run Script ==

To run all nine steps of the equilibration, we will create a run script with MPI. The run script is copied below with the name of the scrit immediately above. 

'''mdequilibration.sh'''

 #!/bin/sh
 #SBATCH --job-name=1s19_equilibration
 #SBATCH --ntasks-per-node=40
 #SBATCH --nodes=2
 #SBATCH --time=8:00:00
 #SBATCH -p long-40core
  
 cd $SLURM_SUBMIT_DIR 
 echo "started Equilibration on 'date' "
 do_parallel="mpirun pmemd.MPI"
  
 parm7="./../03.leap/1s19.wet.complex.parm7"
 coords="./../03.leap/1s19.wet.complex"
  
 MDINPUTS=( 01.min 02.equil 03.min 04.min 05.min 06.equil 07.equil 08.equil 09.equil)
  
 for input in ${MDINPUTS[@]}; do
  
     $do_parallel -O -i ${input}.mdin -o ${input}.mdout -p $parm7 -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r 
 ${input}.rst7 coords=$input
 
 done
  
 echo " Finished equilibration on 'date' "

To run this script:

 sbatch mdequilibration.sh

'''Important Notes'''

We ran into a few problems using this run script sometimes but were easily solved. If:

1) Your equilibration gets stuck at a certain step (ex. cannot get past step 6)                                                                      

Run with pmemd instead of MPI. To change this, delete the '''do_parallel=mpirun pmemd.MPI'''. In the for loop, switch out '''$do_parallel''' with '''pmemd'''.

2) The equilibration will not start, saying that the correct commands are not found.
                                                                                                                  
Try deleting the line '''cd $SLURM_SUBMIT_DIR'''

= Production = 

Create a new directory called '''05.prod''' and change into this directory.

When your equilibration is done running, you should double check that the strucutres look okay after the 09.equil step. Repeat the process as with the examination of the 1s19.wet.complex files in Chimera during the tleap step. If everything looks okay, then you are good to go to continue ot the production runs.

== Input File ==

Below is the input file for '''10.prod.mdin'''

'''For the restraint mask, take out your ligand (so only include protein)

(To do this as an unrestrained simulation, just delete the restraint mask as a whole)

 MD simulations
 &cntrl
 imin=0,           ! Perform MD
 nstlim=5000000,   ! Number of MD steps
 ntx=5,            ! Positions and velocities read formatted
 irest=1,          ! Restart calculation
 ntc=2,            ! SHAKE on for bonds with hydrogen
 dt=0.002,         ! Timestep (ps)
 ntb=2,            ! Constant Pressure
 ntp=1,            ! Isotropic pressure scaling
 barostat=1        ! Berendsen
 taup=0.5          ! Pressure relaxtion time (ps)
 ntf=2,            ! No force evaluation for bonds with hydrogen
 ntt=3,            ! Langevin thermostat
 gamma_ln=2.0      ! Collision Frequency for thermostat
 ig=-1,            ! Random seed for thermostat
 temp0=298.15      ! Simulation temperature (K)
 ntwx= 2500,       ! Write to trajectory file every ntwx steps
 ntpr= 2500,       ! Print to mdout every ntpr steps
 ntwr= 5000000,    ! Write a restart file every ntwr steps
 cut=8.0,          ! Nonbonded cutoff in Angstroms
 ntr=1,            ! Turn on restraints
 restraintmask=":1-433@CA,C,N", ! atoms to be restrained
 restraint_wt=0.1, ! force constant for restraint
 ntxo=1,           ! Write coordinate file in ASCII format
 ioutfm=0,         ! Write trajectory file in ASCII format
 iwrap=1,          ! iwrap is turned on
 /

This step is very similar to the 09.equli.mdin input file. The only things that changed was nstlim, ntc, dt, ntwx, ntpr and ntwr lines.

== Run Script ==

Below is the run script that can be copied into a file called '''mdproduction.sh'''

 #!/bin/sh
 #SBATCH --job-name=1s19_prod
 #SBATCH --ntasks-per-node=24
 #SBATCH --nodes=2
 #SBATCH --time=7-00:00:00
 #SBATCH -p extended-24core
 
 cd $SLURM_SUBMIT_DIR
 echo "started Equilibration on 'date' "
 
 parm7="../03.leap/1s19.wet.complex.parm7"
 coords="../04.equil/09.equil"
 
 MDINPUTS=( 10.prod)
 
 for input in ${MDINPUTS[@]}; do
 
    pmemd -O -i ${input}.mdin -o ${input}.mdout -p ${parm7} -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r 
 ${input}.rst7
 
    coords=$input
 done
 
 echo "Finished Equilibration on `date` "

To run this script:

 sbatch mdproduction.sh

= Analysis =
Now that we have created the MD trajectory, we want to run an analysis of how the ligand-receptor system has changed and what interactions are critical for ligand binding. We will do this by measuring RMSD, H-bonds, and MM-GBSA.

== RMSD ==
Root-mean-square deviations depict how far the ligand and receptor have moved from their starting position. We will begin by creating the following input files:
 touch cpptraj_rmsd_lig.in, cpptraj_rmsd_rec.in, cpptraj_strip_wat.in

First we will strip the trajectory of all waters and ions.
 
 vim cpptraj_strip_wat.in

 #!/usr/bin/sh
 parm ../003.tleap/1s19.wet.complex.parm7
 #read in trajectory
 trajin ../004.productionrun/10.prod.trj
 #read in reference
 reference ../003.tleap/1s19.wet.complex.rst7
 #compute RMSD + align CA to crystal structure
 rmsd rms1 reference :1-263@CA
 #strip solvent
 strip :WAT:Na+:Cl-
 #create gas-phase trajectory
 trajout 1S19_stripfit_restrained_gas.trj nobox

 cpptraj -i cpptraj_strip_wat.in

Then we will measure the RMSD of the ligand and receptor respectively.
 
 vim cpptraj_rmsd_lig.in

 #!/usr/bin/sh
 #trajin the trajectory
 trajin 1S19_stripfit_restrained_gas.trj
 #read in reference
 reference ../003.tleap/1s19.gas.complex.rst7
 #compute RMSD (do not fit internal geometris first, included rigid body motions, convert frames to ns (framenum*.005)
 rmsd rms1 ":254&!(@H=)" nofit mass out 1S19_lig_restrained_rmsd_nofit.dat time .005
 #histogram the nofit rmsd
 histogram rms1,*,*,.1,* norm out 1S19_lig_restrained_rmsd_nofit_histogram.dat

'''Make sure to change the number of the ligand in the "rmsd rms1" line to match your particular system'''
To submit the job:

 cpptraj -p ../003.tleap/1s19.gas.complex.parm7 -i cpptraj_rmsd_lig.in

 vim cpptraj_rmsd_rec.in

 #!/usr/bin/sh
 #trajin the trajectory
 trajin 1S19_stripfit_restrained_gas.trj
 #read in reference
 reference ../003.tleap/1s19.gas.complex.rst7
 #compute RMSD (do not fit internal geometries first, included rigid body motions, convert frames to ns (framenum*.005)
 rmsd rms1 ":1-253&!(@H=)" nofit mass out 1S19_rec_restrained_rmsd_nofit.dat time .005
 #histogram the nofit rmsd
 histogram rms1,*,*,.1,* norm out 1S19_rec_restrained_rmsd_nofit_histogram.dat

'''Make sure to change the residues of the protein in the "rmsd rms1" line to match your particular system'''
To submit the job:

 cpptraj -p ../003.tleap/1s19.gas.complex.parm7 -i cpptraj_rmsd_rec.in

You should get an output that looks like the one below.

[[File:1s19_rmsd.png|thumb|center|400px| Figure 1. Ligand and Receptor rmsd values]]

== Hydrogen Bonds ==

Here we want to figure out what hydrogen bonds our ligand forms with our receptor. We will begin by creating an input file:

 gedit cpptraj_hbond.in

 !/usr/bin/sh
 #read in trajectory
 trajin ../004.productionrun/10_prod.trj
 #wrap everything in one periodic cell - could cause problems, may comment out #autoimage if problems later
 autoimage
 #compute intra + water-mediated H-bonds
 hbond hb1 :1-263 out 1S19_calcipotriol_hbond.out avgout 1S19_calcipotriol_hbond_avg.dat solventdonor :WAT solventacceptor :WAT@-O
 nointramol brid\
 geout 1S19_calcipotriol_bridge-water.dat dist 3.0 angle 140

Run this input file using the same cpptraj tool:

 cpptraj -p ../002.tleap_build/1S19_wetcomplex.parm7 -i cpptraj_hbond.in

== MMGBSA ==

Molecular Mechanics-Generalized Born Solvent Accessibility is a method to estimate the free energy of binding of our ligand and receptor by evaluating the free energy of unsolvated and solvated ligand and receptor separately then combining them. To measure this binding, we will first create an input file:

 gedit mmgbsa.in

 mmgbsa 1S19 analysis
 &general
 	interval=1, netcdf=1,
 	keep_files=0,
 /
 &gb
 	igb=8
 	saltcon=0.0, surften=0.0072,
 	surfoff=0.0, molsurf=0,
 /
 &nmode
 	drms=0.001, maxcyc=10000,
 	nminterval=250, nmendframe=2000,
 	nmode_igb=1,
 /

We need to create a SLURM script to run this input file. Create the following file '''mmgbsa_1S19_slurm.sh''':

 #bin/bash
 #SBATCH --time=2-00:00:00
 #SBATCH --nodes=2
 #SBATCH --ntasks=28
 #SBATCH --job-name=1S19_MMGBSA
 #SBATCH --output=1S19_MMGBSA.log
 #SBATCH -p extended-28core
 #Define topology files
 solv_parm="../002.tleap_build/1S19_wetcomplex.parm7"
 complex_parm="../002.tleap_build/1S19_gascomplex.parm7"
 receptor_parm="../002.tleap_build/1S19_gasrec.parm7"
 lig_parm="../002.tleap_build/1S19_gaslig.parm7"
 trajectory="../004.productionrun/10_prod.trj"
 MMGBSA.py -O -i mmgbsa.in \
 	-o 1S19_mmgbsa_results.dat \
 	-eo 1S19_mmgbsa_perframe.dat \
 	-sp ${solv_parm} \
 	-cp ${complex_parm} \
 	-rp ${receptor_parm} \
 	-lp ${lig_parm} \
 	-y ${trajectory}