Revision as of 17:54, 5 May 2024

Introduction

AMBER is a molecular dynamics program that simulate the behavior of a protein/ligand complex of interest. AMBER considers all atoms and models their interactions over time, continuously updating positions and forces in small time increments. For this tutorial, we will focus on 2ITO.

Getting Started

Update the directory structure to the following:

Structure

Similarly to the DOCK tutorial, we need to obtain the structure of the protein and ligand. For the protein, we want to fill in all loops even if it is not near the binding pocket, since AMBER considers all atoms in its calculations. Follow the DOCK tutorial for 2ITO to do this.

For the protein, do not add H or add charge. Save as 2ITO_protein_only_for_AMBER.pdb and 2ITO_protein_only_for_AMBER.mol2. For the ligand, add H and add charge. Save as 2ITO_ligand_only_for_AMBER.pdb and 2ITO_ligand_only_for_AMBER.mol2.

Force Field Parameters

Force field parameters are required to calculation the interactions between all atoms. This needs to be done for the ligand. Create the following slurm script as follows:

 vi 2ITO_ffp.slurm

and have the following within the file:

Make sure to change LIG -nc 0 based off of your ligand. It is the charge on the ligand. For 2ITO, this charge is +1.

Once the slurm file has run, the following will show up in your directory:

 2ITO_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

2ITO_ligand_antechamber.mol2 is the file with the parameters we generated.

In the command line, load amber 16:

 module load amber/16

Run the following command to modify the parameters slightly:

parmchk2 -i 2ITO_ligand_antechamber.mol2 -f mol2 -o 2ITO_ligand.am1bcc.frcmod

You will see 2ITO_ligand.am1bcc.frcmod file in your directory.

TLeap

TLeap is a program with AMBER that will prepare the system for MD simulations. It will generate the gas-phase and solvated systems.

Create the file:

 vi 2ITO_tleap.in

and add the following to the file:

Create the following slurm file:

 vi 2ITO_tleap.slurm

and add the following to the file:

Run the slurm and you should get the following new files in your directory:

 2ITO.complex.parm7    
 2ITO.gas.complex.rst7  
 2ITO.gas.ligand.rst7     
 2ITO.gas.receptor.rst7      
 leap.log
 2ITO.gas.complex.pdb  
 2ITO.gas.ligand.parm7  
 2ITO.gas.receptor.parm7       
 2ITO.wet.complex.pdb  
 2ITO.wet.complex.prmtop
 2ITO.wet.complex.rst7    
 tleap_output.txt

You can look at the files you created by moving the files to your local computer. Move 2ITO.wet.complex.rst7 and 2ITO.wet.complex.prmtop. Open chimera and follow the following commands:

 Tools → MD/Ensemble Analysis → MD Movie

Click browse and put in the prmtop file. Add the rst7 trajectory file. You should be able to view your system.

Equilibration

Look at the 2ITO.gas.complex.pdb file that was just generated. Scroll all the way down to see the residues in the complex. In our example, the protein is residues 1-324 and the ligand is 325.

We need to equilibrate the system before running the MD simulations. Note, for files 1-7, set the restraint mask to the entire system (1-325). For files 8-9, set the restraint mask to cover only the receptor (1-324).

 vi 01.min.mdin
 vi 02.equil.mdin
 vi 03.min.mdin
 vi 04.min.mdin
 vi 05.min.mdin
 vi 06.equil.mdin
 vi 07.equil.mdin
 vi 08.equil.mdin
 vi 09.equil.mdin

Production

The system is all equilibrated and now we can run production run.

 vi 10.prod.mdin

and add the following:

Create the slurm file:

 production.slurm

and add the following:

Run the slurm script

Analysis

Now we can analyze the simulation by looking at the RMSD, H-Bonds, and MM/GBSA results.

RMSD

First we want to strip all the water:

vi cpp_strip_water.in

with the following:

To look at the RMSD of the ligand, we only want to look at the residue that includes the ligand:

vi cpp_rmsd_lig.in

with the following:

Run it with the following command:

 cpptraj -i cpp_strip_water.in

Run it with the following command:

 cpptraj -p ../003_tleap/2ITO.complex.parm7 -i cpp_rmsd_lig.in

Now we want to look at the RMSD of the receptor. Include the residues that include only the receptor:

vi cpp_rmsd_receptor.in

with the following:

Run it with the following command:

 cpptraj -p ../003_tleap/2ITO.complex.parm7 -i cpp_rmsd_receptor.in

If you want to look at the histogram, you can graph it using python.

H-Bonds

To look at the hydrogen bonds that occur in the system during the simulation, make sure to include the receptor and ligand in the mask. Create the program:

 vi cpp_hbonds.in

with the following:

Run it with the following command:

 cpptraj -p ../003_tleap/2ITO.wet.complex.prmtop -i cpp_hbonds.in

MM/GBSA

Now we will calcualte the free energy of the system. Create the program:

 vi mmgbsa_2ITO.in

with the following:

and run.