Revision as of 19:16, 1 April 2021

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 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 use will be the wet complex files.

Equilibration

Input Files

Run Script

Produduction

Input File

Run Script

Analysis

RMSD

Hydrogen Bonds

MMGBSA