Revision as of 14:33, 20 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.

 Tools -> General Controls -> Command Line

on the command line, type:

 open 4sov

then:

 Select -> Structure -> protein

which will select the receptor, and then go to

 Select -> Invert (all models)

to select everything other than the receptor in the file. Then, go to

 Actions -> Atoms/Bonds -> delete

which will isolate the receptor.

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 as 4s0v_nolig_noH_nocharge.mol2. This will save a mol2 of just the protein, without hydrogens or charges added.

Alternatively, you can follow the steps in the *[1] tutorial to do this. The inputs we need are the isolated protein with NO hydrogens and NO charges; and the ligand with hydrogens and charges. In other words, once you isolate the protein structure in Chimera, save it with a filename such as, 4s0v_protein_for_AMBER.pdb.

Then isolate the ligand structure, add hydrogens and re-do whatever protonation changes you made in the *[2] tutorial.

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

We save this ligand file (with hydrogens) as: 4s0v_ligand_only.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.

Generate a 002.parameters directory and cd into it.

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.

 mkdir 003.tleap

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):

 nano 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-96core
 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.

 mkdir 004.equil

Nine input files are required for system equilibration.

These files fall into two types: min.mdin - will minimize only hydrogen atoms equil.mdin - will minimize system including non-hydrogen atoms, depending upon the restraint mask for atoms involved

What are restraints? "!" means all atoms except those that follow. "@H=" means all hydrogen atoms.

Here, we want to have restraints on all protein and ligand atoms

What are restraint weights? Check the AMBER handbook for a better description, but in brief, corresponds to Hooke's law and the energy penalty associated with motion

Create 01.min.mdin

 nano 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
  /

Create 02.equil.mdin

 nano 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

 nano 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

nano 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

 nano 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

 nano 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

 nano 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

 nano 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
 /

In the above file you will see a line:

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

The number 490 needs to be updated for your specific system. This is done by opening the 4s0v.wet.complex.pdb file in your 003.tleap directory and searching for atom 'LIG'. You will see something similar to:

The number shown for your system is what needs to be put in 08.equil.mdin.

Create 09.equil.mdin.

 nano 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
 /

Once again you will see a line:

 restraintmask=":1-489@CA,C,N", ! atoms to be restrained

This number is simply one less than the number used in 08.equil.mdin. Make sure to put the appropriate number for your system in your file.

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 37 new files including a file named logfile. Check to file for errors. If there are none, you can move onto the next step.

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 your 005.production_run directory.

Create the input file, 10.prod.min

 vi 10.prod.min

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

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.

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

#!/bin/bash
#
#SBATCH --job-name=4s0v_production
#SBATCH --output=production.txt
#SBATCH --ntasks-per-node=24
#SBATCH --nodes=3
#SBATCH --time=48:00:00
#SBATCH -p long-24core 

module load amber/16 

do_parallel="/gpfs/software/intel/parallel-studio-xe/2018_3/compilers_and_libraries/linux/mpi/bin64/mpirun -np 80 /gpfs/software/amber/16/intel/cpu/bin/pmemd.MPI"

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

Unrest Analysis