Latest revision as of 14:57, 29 March 2023

Introduction

AMBER is a molecular dynamics program that can be run on your protein/ligand complex to ensure that the interactions between the two structures are stable. DOCK shows us how the two interact with each other at one point in time. AMBER looks at those interactions over time to ensure that forces will not occur which will push the ligand out of the binding site as the complex naturally moves. This tutorial will again be working with PDB #4s0v

Setting Up Your Environment

Just as with DOCK you should set up for directory structure at this point to keep everything organized and easy to find. We will be creating a new structure which looks like:

Structure

Before starting the analysis it's best to download a new protein/ligand complex from the PDB and isolate both the protein and ligand structures. This time we will be using the command line function in Chimera. To open the command line go to:

 Tools -> General Controls -> Command Line

on the command line, type:

 open 4s0v

Once the file is opened, we will move back to using the drop down menus. Choose:

 Select -> Structure -> protein

which will select the protein. Choose:

 Select -> Invert (all models)

This will change your selection to everything other than the protein. To delete the current selection, choose:

 Actions -> Atoms/Bonds -> delete

This will delete everything from the session except the protein structure. Save this file under a new name, 4s0v_protein_only_for_AMBER.pdb.

For the protein file, it’s important to model any missing loops before running AMBER on the complex. You might not have done this for the docking tutorial because the loops were far enough from the binding site to not matter but for this step it needs to be done. After adding in any missing loops with

 Tools -> Structure Editing -> Model/Refine Loops

Go to

 File -> Save Mol2... 

and save the file under a new name, such as 4s0v_protein_only_no_charges.mol2.

You then need to follow the same steps above but this time isolating the ligand and not the protein. Once you have the ligand in its own file, 4s0v_ligand_only_for_AMBER.pdb, you need to make whatever protonation changes you made when preparing the ligand for docking. See *[1] for these steps.

For our purposes in this tutorial, we will leave the ligand as a .pdb for parametrization, since defining the connectivity of the oxygens where we removed hydrogens can be tricky. However, if you are following this for your own ligands, you can explicitly define the charges if you are confident.

This file can now be saved as 4s0v_ligand_only_charges_for_AMBER.pdb

Once these two files have been generated, scp them over to the 001.structure directory on Seawulf.

Force Field Parameters

AMBER needs force field parameters to run correctly. This only needs to be done for the ligand. These commands will be done on the command line again, please cd into your 002.forceFieldParameters directory. To generate ligand parameters execute the following slurm script:

 #!/bin/bash
 #
 #SBATCH --job-name=4s0v_AMBER_parameters
 #SBATCH --output=parameters_output.txt
 #SBATCH --ntasks-per-node=24
 #SBATCH --nodes=6
 #SBATCH --time=48:00:00
 #SBATCH -p long-24core
 
 module load amber/16
 
 antechamber -i ../001.structure/4s0v_ligand_only.pdb -fi pdb -o 4s0v_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0

The important parameter in the above command is the -nc option at the end. This is telling antechamber what the total charge is on your ligand. This number needs to be the same as the number used in the previous step when you added charges to the structure in Chimera. In our case, the total charge on the ligand should be 0.

Once this has completed running you will see multiple new files in your directory including, specifically:

 4s0v_ligand_antechamber.mol2  
 ANTECHAMBER_AC.AC0     
 ANTECHAMBER_AM1BCC_PRE.AC  
 ANTECHAMBER_BOND_TYPE.AC0  
 sqm.in   
 sqm.pdb
 ANTECHAMBER_AC.AC             
 ANTECHAMBER_AM1BCC.AC  
 ANTECHAMBER_BOND_TYPE.AC   
 ATOMTYPE.INF               
 sqm.out

4s0v_ligand_antechamber.mol2 is the file with the parameters we just generated.

Now that we have defined parameters for the ligand, we need to modify our force field parameters slightly. Run the command:

 parmchk2 -i 4s0v_ligand_antechamber.mol2 -f mol2 -o 4s0v_ligand.am1bcc.frcmod

If you get an error when running this line, try typing:

 module load amber/16

and then running the command again. Once it's done running you will see the 4s0v_ligand.am1bcc.frcmod file in your directory.

TLeap Implemenation

The goal of molecular dynamics simulations is to model the behavior of molecular systems and observe the behavior and interactions between constituents in those systems. Here, we investigate the interactions between our ligand and our receptor. Our MD analysis using AMBER will allow is to experimentally model this interactions over a given timeframe, and from these simulations we can estimate the affinity of the receptor for this ligand. We will be working on the command line so please cd into your 003.tleap directory.

We will first generate the gas-phase and solvated systems, and neutralize both systems.

To create the input script (which should be run on the cluster, not on a login node, see below):

 vi 4s0v_tleap.in

And then add:

 #!/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/4s0v_built.pdb
  
 #THIS IS WHERE YOU WOULD DEFINE DISULFIDE BONDS
 #NUMBERING SHOULD MATCH INPUT PDB FILE
 #bond rec.649.SG rec.762.SG
 #bond rec.454.SG rec.472.SG
 #bond rec.444.SG rec.447.SG
 #bond rec.328.SG rec.339.SG
 #bond rec.385.SG rec.394.SG
 ###load ligand frcmod/mol2
 loadamberparams ../002.parameters/4s0v_ligand.am1bcc.frcmod
 lig=loadmol2 ../002.parameters/4s0v_ligand_antechamber.mol2
  
 ###create gase-phase complex
 gascomplex= combine {rec lig}
  
 ###write gas-phase pdb
 savepdb gascomplex 4s0v.gas.complex.pdb
 
 ###write gase-phase toplogy and coord files for MMGBSA calc
 saveamberparm gascomplex 4s0v.complex.parm7 4s0v.gas.complex.rst7
 saveamberparm rec 4s0v.gas.receptor.parm7 4s0v.gas.receptor.rst7
 saveamberparm lig 4s0v.gas.ligand.parm7 4s0v.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 4s0v.wet.complex.pdb
  
 ###check the system
 charge solvcomplex
 check solvcomplex
  
 ###write solvated toplogy and coordinate file
 saveamberparm solvcomplex 4s0v.wet.complex.prmtop 4s0v.wet.complex.rst7
 quit

This input file should be executed on the cluster, not in a login node. To do so, create and run the following script: 4s0v_tleap.slurm.

 #!/bin/bash
 #
 #SBATCH --job-name=4s0v_tleap
 #SBATCH --output=tleap_output.txt
 #SBATCH --ntasks-per-node=24
 #SBATCH --nodes=6
 #SBATCH --time=48:00:00
 #SBATCH -p long-24core
 module load amber/16
 tleap -f 4s0v_tleap.in

Execute this script with:

 sbatch 4s0v_tleap.slurm

When this is done running you will see multiple files in your directory. Specifically:

 4s0v.complex.parm7    
 4s0v.gas.complex.rst7  
 4s0v.gas.ligand.rst7     
 4s0v.gas.receptor.rst7  
 4s0v_tleap.slurm      
 4s0v.wet.complex.prmtop  
 leap.log
 4s0v.gas.complex.pdb  
 4s0v.gas.ligand.parm7  
 4s0v.gas.receptor.parm7  
 4s0v_tleap.in           
 4s0v.wet.complex.pdb  
 4s0v.wet.complex.prmtop
 4s0v.wet.complex.rst7    
 tleap_output.txt

Scp 4s0v.wet.complex.rst7 and 4s0v.wet.complex.prmtop to your local computer.

In Chimera, open using:

 Tools → MD/Ensemble Analysis → MD Movie

In the prmtop section, select 4s0v.wet.complex.prmtop, and then hit "Add" and select the 4s0v.wet.complex.rst7 file. Your receptor should now open in Chimera.

Everything looks good so we can move onto the next step.

Complex Equilibration

Before we can run our structure through the AMBER program we need to minimize it and make sure it's at equilibrium. These commands will again be done on the command line so please cd into your 004.equil directory.

Nine input files are required for system equilibration and AMBER will process them in order. You can imagine these steps as 'heat-treating' your complex - you apply heat to the system and then let it relax - and repeat until the system is at rest. Each of these nine files is one step in this process. For each step there are two files:

min.mdin - will minimize only hydrogen atoms equil.mdin - will minimize system including non-hydrogen atoms, depending upon the restraint mask for atoms involved

These input files will also direct AMBER on which atoms to restrain during equilibration. There a specific line in each file:

 restraintmask

after that command, if you see a "!", that is telling AMBER to exclude any atoms that follow; if you see "@H=", that is telling AMBER to restrain all the hydrogen atoms. In the first 7 input files we want to restrain all residues in our complex, including the ligand. For the last two steps we will only restrain the protein and not the ligand.

There is also a line:

 restraint_wt

What are restraint weights? Check the AMBER handbook for a better description, but in brief, this is a constant similar to a spring constant used in Hooke's law. For the first files we use a larger restraint_wt and a lower constant for the final files. Before we can create any input files we need to know how many residues are in our complex. Do this by looking at at the 4s0v.gas.complex.pdb file created in the previous step and looking at the last line:

We see that there are 490 residues in our complex. 489 in the protein and one for the ligand.

Create 01.min.mdin

 vi 01.min.mdin

Copy the following lines:

  Minmize all the hydrogens
  &cntrl
  imin=1,           ! Minimize the initial structure
  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=":1-490 & !@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
  /

In the above input file you'll see a line:

 restraintmask=":1-490@CA,C,N", ! atoms to be restrained.

This is where the number of residues for your system needs to be updated. This change needs to be done for the following input files as well.

Create 02.equil.mdin

 vi 02.equil.mdin

Copy the following lines:

  MD simualation
  &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=":1-490 & !@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
  /

Create 03.min.mdin

 vi 03.min.mdin

Copy the following lines:

  Minmize 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=":1-490 & !@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
  /

Create 04.min.mdin

vi 04.min.mdin

Copy the following lines:

 Minmize 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=":1-490 & !@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
 /

Create 05.min.mdin

 vi 05.min.mdin

Copy the following lines:

 Minmize 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=":1-490 & !@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
 /

Create 06.equil.mdin

 vi 06.equil.mdin

Copy the following lines:

 MD simualation
 &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=":1-490 & !@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
 /

Create 07.equil.mdin

 vi 07.equil.mdin

Copy the following lines:

 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=":1-490 & !@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
 /

Create 08.equil.mdin

 vi 08.equil.mdin

Copy the following lines:

 MD simulations
 &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=":1-489@CA.C.N", ! atoms to be restrained, only the backbone
 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
 /

NOTE: for the above input file we only want to restrain the protein and not the ligand so the '490' needs to be change to '489'.

Create 09.equil.mdin.

 vi 09.equil.mdin

Copy the following lines:

 MD simulations
 &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=":1-489@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
 /

NOTE: for the above input file we only want to restrain the protein and not the ligand so the '490' needs to be change to '489'.

Once you have all nine files in your directory we need to create a slurm script, 4s0v.equil.slurm, to run them:

 #!/bin/bash
 #
 #SBATCH --job-name=4s0v_equilibration
 #SBATCH --output=equilibration_output.txt
 #SBATCH --ntasks-per-node=24
 #SBATCH --nodes=1
 #SBATCH --time=48:00:00
 #SBATCH -p long-24core
 
 module load amber/16
 
 do_parallel="mpirun -np 80 pmemd.MPI"
 
 prmtop="../003.tleap/4s0v.wet.complex.prmtop"
 coords="../003.tleap/4s0v.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 $prmtop -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r ${input}.rst7
   coords=$input
 done

Submit the above file to Seawulf by typing:

 sbatch 4s0v.equil.slurm

Once the program finishes you will see multiple new files including a file named logfile. Check to file for errors. If there are none, you can move onto the next step. You should also check the .trj files from each step to make sure that the system appears rational. Below is the equillibrated system following 09.equil.mdin

Production Run

Now that our system is set up properly we can move onto an MD production run of the structure. This will again be completed on the command line, please cd into 005.production

Create the input file, 10.prod.min

 nano 10.prod.mdin

And copy the following lines:

  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=":1-489@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
  $end

Note for the restraintmask line, use the same number as for the 09.equil.mdin file in the previous step. In our case, this number is 489.

 nano production.sh

This input file can now be run on Seawulf using the following slurm script:

 #!/bin/bash
 #
 #SBATCH --job-name=4s0v_production
 #SBATCH --output=4s0v_production.txt
 #SBATCH --ntasks-per-node=28
 #SBATCH --nodes=4
 #SBATCH --time=48:00:00
 #SBATCH -p long-28core 

 module load amber/16

 do_parallel="mpirun pmemd.MPI"

 prmtop="../003.tleap/4s0v.wet.complex.prmtop"
 coords="../004.equil/09.equil"

 MDINPUTS=(10.prod)

 for input in ${MDINPUTS[@]}; do
   $do_parallel -O -i ${input}.mdin -o ${input}.mdout -p $prmtop -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r ${input}.rst7
   coords=$input
 done

Analysis

The output of the production run performed in the previous step is to generate and MD trajectory of the ligand/protein complex. Once this is done we need to analyze the results. We will analysis three different aspects of the complex:

• RMDS (the distance the ligand and protein have moved from their initial positions)
• H-bonds (what hydrogren bonds have been formed between the ligand and protein)
• MM-GBSA (estimation of the free binding energy between the ligand and protein)

We will again be working on the command line, please cd into your 006a.RMSD directory

RMSD

To perform the RMSD calculations, first create an input file which will delete the water and charges from the production trajectory.

 nano cpp_strip_water.in

with the following inputs. Note that this should cover all CA atoms (including the ligand) in your complex

 #!/usr/bin/sh
 parm ../003.tleap/4s0v.wet.complex.prmtop
 #read in trajectory
 trajin ../005.production/10.prod.trj
 #read in reference
 reference ../003.tleap/4s0v.wet.complex.rst7
 #compute RMSD + align CA to crystal structure
 rmsd rms1 reference :1-490@CA
 #strip solvent
 strip :WAT:Na+:Cl-
 #create gas-phase trajectory
 trajout 4s0v_stripfit_restrained_gas.trj nobox
 cpp_strip_water.in (END)

The reference mask should contain BOTH the ligand and receptor in its value.

Now run this file using the following command:

 cpptraj -i cpp_strip_water.in

Next, create another input file which will be used to calculate the RMSD of the ligand by typing

 nano cpp_rmsd_lig.in

with inputs

 #!/usr/bin/sh
 #trajin the trajectory
 trajin 4s0v_stripfit_restrained_gas.trj
 #read in reference
 reference ../003.tleap/4s0v.gas.complex.rst7
 #compute RMSD (do not fit internal geometris first, included rigid body motions, convert frames to ns (framenum*.005)
 rmsd rms1 ":490&!(@H=)" nofit mass out 4s0v_lig_restrained_rmsd_nofit.dat time .005
 #histogram the nofit rmsd
 histogram rms1,*,*,.1,* norm out 4s0v_lig_restrained_rmsd_nofit_histogram.dat 

And check that the reference mask only factors in the ligand (490) in this file. Run this input file using the following command:

 cpptraj -p ../003.tleap/4s0v.complex.parm7 -i cpp_rmsd_lig.in

Next, the receptor RMSD can be calculated by creating the following input file:

 nano cpp_rmsd_receptor.in

with inputs

 #!/usr/bin/sh
 #trajin the trajectory
 trajin 4s0v_stripfit_restrained_gas.trj
 #read in reference
 reference ../003.tleap/4s0v.gas.complex.rst7
 #compute RMSD (do not fit internal geometries first, included rigid body motions, convert frames to ns (framenum*.005)
 rmsd rms1 ":1-489&!(@H=)" nofit mass out 4s0v_receptor_restrained_rmsd_nofit.dat time .005
 #histogram the nofit rmsd
 histogram rms1,*,*,.1,* norm out 4s0v_receptor_restrained_rmsd_nofit_histogram.dat

And check that the reference mask only takes into account the receptor (1-489). Now run this file using the following command:

 cpptraj -p ../003.tleap/4s0v.complex.parm7 -i cpp_rmsd_receptor.in

The output files from these analyses will be:

 4s0v_lig_restrained_rmsd_nofit.dat            
 4s0v_receptor_restrained_rmsd_nofit_histogram.dat  
 4s0v_lig_restrained_rmsd_nofit_histogram.dat  
 4s0v_stripfit_restrained_gas.trj
 4s0v_receptor_restrained_rmsd_nofit.dat

Examine the histogram.dat files, or plot if desired, to note the change in the RMSD of the ligand (or the receptor) over the course of the simulation. Since this was docked previously, we would hope that it would stay under 2 angstroms over the course of the simulation. There may be other simulations were movement is acceptable (or even desired) but this is not that case.

H-bonds

Next, the number of hydrogen bonds will be analyzed, including what these bonds are. Create the following input file:

 nano cpp_hbonds.in

with the following:

!/usr/bin/sh
#read in trajectory
trajin ../004.production/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 :1-490 out 4s0v_hbond.out avgout 4s0v_hbond_avg.dat solventdonor :WAT solventacceptor :WAT@-O
nointramol brid\
geout 4s0v_bridge-water.dat dist 3.0 angle 140

Be sure that the hbobnd hb1 parameter corresponds to the residue numbers of the relevant ligand AND receptor (490 in our case). Run with:

cpptraj -p ../003.tleap/4s0v.wet.complex.prmtop -i cpp_hbonds.in

MM-GBSA Analysis

Finally, we will calculate free energies of binding in this system with MM-GBSA. Create the following input file:

 nano mmgbsa_4s0v.in

This file should contain the following:

mmgbsa 4s0v 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,
 /

Since this may take a while to run we should execute the script using Seawulf. Generate the following script:

 4s0v_mmgbsa.sh

With the following as contents:

 #bin/bash
 #SBATCH --time=2-00:00:00
 #SBATCH --nodes=2
 #SBATCH --ntasks=28
 #SBATCH --job-name=4s0v_MMGBSA
 #SBATCH --output=4s0v_MMGBSA.log
 #SBATCH -p extended-28core
 #Define topology files
 solv_parm="../003.tleap/4s0v.wet.complex.prmtop"
 complex_parm="../003.tleap/4s0v.complex.parm7"
 receptor_parm="../003.tleap/4s0v.gas.receptor.parm7"
 lig_parm="../003.tleap/4s0v.gas.ligand.parm7"
 trajectory="../005.production/10_prod.trj"
 MMPBSA.py -O -i mmgbsa.in \
      -o 4s0v_mmgbsa_results.dat \ 
      -eo 4s0v_mmgbsa_perframe.dat \
      -sp ${solv_parm} \
      -cp ${complex_parm} \
      -rp ${receptor_parm} \
      -lp ${lig_parm} \
       -y ${trajectory}

Execute this with:

 sbatch 4s0v_mmgbsa.sh