2023 AMBER tutorial 3 with PDBID 2P16

Introduction

Howdy! If you've gotten this far with your MD project, you've now moved out of the realm of DOCK6 and into the world of molecular dynamics (MD). This tutorial will cover how to use the AMBER 16 software package to simulate your protein-ligand complex on a sub-microsecond timeframe and use the resulting data to calculate binding energies.

Preparing the Structure

Before you start any preparation, you need to keep yourself organized to make sure that you're running the right files and jobs for your MD simulation, otherwise, it may explode.

To do this, you can either copy the example AMBER directory to your scratch directory (this may change per year):

/gpfs/projects/AMS536/2023

Or, you can create the following directories:

mkdir 000.parameters
mkdir 001.tleap_build
mkdir 002.equilibration
mkdir 003.production
mkdir 004.analysis
mkdir zzz.master

For this tutorial, we will be copying the example AMBER setup into our folder to run our complex.

Simulation Parameters

If you're looking at this tutorial after the DOCK6 tutorial and are looking to use the original ligand, then you may want to fetch a fresh PDB file for this. Otherwise, make sure you Cartesian minimize any new ligands before sending them through the gauntlet that is MD simulation and preparation.

All of your original (stock/DOCK6 generated) files should be put into the zzz.master folder, but make sure you're in the proper directory for each step!

Receptor File Generation

To generate a fresh copy of the receptor file, go to the Protein Data Bank (PDB) website and download the PDB structure for 2P16, 2p16.pdb, which will contain the ligand-receptor complex. Open the pdb file in Chimera and go to

 Select -> Structure -> Main

Alternatively, you can instead for this protein:

 Select -> Residues -> All Standard Residues

which will select the receptor protein, and then:

 Select -> Invert (all models)

to select everything except the receptor. These will all be deleted:

 Actions -> Atoms/Bonds -> delete

Now, save your receptor as a PDB. You can save it as a mol2, but you are much more likely to bump into problems down the road:

 File -> Save PDB... 

and save as 2P16_rec.pdb. Close Chimera. You may have thought you were done, but there are still issues with your protein. Specifically, AMBER will not read disulfide bonds between cysteines properly. To fix this, you will need to manually edit your PDB file (using Notepad, vi, nano, etc.) and edit all disulfide-bonded cysteine by changing the residue from CYS to CYX. In addition, there will be some lines to add to your tleap.build.in file later on to make sure that AMBER properly recognizes the disulfide bonds. Save these changes.

Ligand File Generation

To generate the ligand file, open the 2P16.pdb file again, and go to

 Select -> Structure -> Main

Alternatively, you can instead for this protein:

 Select -> Residues -> All Standard Residues

Then you will delete the protein:

 Actions -> Atoms/Bonds -> delete

You should also delete all non-essential cofactors, ions, and waters:

 Select -> Residue -> HOH -> Actions -> Atoms/Bonds -> delete

 Select -> Residue -> CA -> Actions -> Atoms/Bonds -> delete

to delete everything except for the GG2 residue, which is the ligand. After this is completed, you will be adding hydrogens and partial charges:

 Tools > Structure editing > DockPrep

Make sure to select the same parameters as in the DOCK6 prep (ff14SB and AM1BCC). Save this file:

 File -> Save Mol2... 

and save as 2P16_lig_withH.mol2. Close Chimera. Transfer them back into your Seawulf directory (./zzz.master). The next step involved getting your ligands ready for tleap.

Force Field Parameterization

Before running TLeap, we need to generate appropriate force field parameters for the ligand. You can achieve this using antechamber- a program built specifically for small molecules, followed by parmchk to verify if it ran correctly. Make sure that the -nc flag corresponds to the net charge of the molecule.

If your ligand happens to be a peptide, you can skip this step.

Run the following commands:

antechamber -i ../zzz.master/2p16_lig_wH.mol2 -fi pdb -o 2p16_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0
parmchk2 -i 2p16_ligand_antechamber.mol2 -f mol2 -o 2p16_ligand.am1bcc.frcmod

TLeap

TLeap will generate parameter and topology files for the protein and ligand complex. Switch to the 001.tleap_build directory and open a file names tleap.build.in and type the following: