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| − | =Introduction=
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| − | 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
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| − | ==Setting Up Your Environment==
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| − | 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:
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| − | =Structure=
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| − | 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. Follow the steps in the *[http://ringo.ams.stonybrook.edu/index.php/2023_DOCK_tutorial_1_with_PDBID_4S0V] 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 *[http://ringo.ams.stonybrook.edu/index.php/2023_DOCK_tutorial_1_with_PDBID_4S0V] tutorial. Once the protonation state is correct, add charges and save the file as 4s0v_ligand_for_AMBER.mol2.
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| − | Once these two files have been generated, scp them over to the 001.structure directory on Seawulf.
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| − | =Force Field Parameters=
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| − | AMBER needs force field parameters to run correctly. This only needs to be done for the ligand since information about the protein has been done over previous years. To generate the necessary files for AMBER we will be working on the command line on Seawulf, please cd into your 002.forceFieldParameters directory.
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| − | To generate the parameters for the ligand we need to run the following slurm script:
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| − | #!/bin/bash
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| − | #
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| − | #SBATCH --job-name=4s0v_AMBER_parameters
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| − | #SBATCH --output=parameters_output.txt
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| − | #SBATCH --ntasks-per-node=24
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| − | #SBATCH --nodes=6
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| − | #SBATCH --time=48:00:00
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| − | #SBATCH -p long-24core
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| − | module load amber/16
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| − | antechamber -i ../001.structure/4s0v_ligand_for_AMBER.pdb -fi pdb -o 4s0v_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0
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| − | 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.
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| − | Once this has completed running you will see multiple new files in your directory including, 4s0v_ligand_antechamber.mol2. This is the file with the parameters we just generated and what we need to use to generate the next file we need, a frcmod file, which will contain modified force field parameters. To generate the frcmod file, run the command:
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| − | parmchk2 -i 4s0v_ligand_antechamber.mol2 -f mol2 -o 4s0v_ligand.am1bcc.frcmod
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| − | If you get an error when running this line, try typing:
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| − | module load amber/16
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| − | and then running the command again. Once it's done running you will see the 4s0v_ligand.am1bcc.frcmod file in your directory.
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| − | =TLeap Implemenation=
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| − | Remembering that the point of running AMBER on our complex is to investigate the dynamics of the entire complex over time. In this next step we bring together the separate ligand and protein files so AMBER can be run on the whole comlex. This is done with a program called tleap. Again we will be working on the command line in Seawulf, please cd into your 003.tleap directory.
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| − | To create the input file:
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| − | vi 4s0v_tleap.in
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| − | and type the following lines into the input file:
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| − | #!/usr/bin/sh
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| − | ###load protein force field
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| − | source leaprc.protein.ff14SB
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| − | ###load GAFF force field (for our ligand)
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| − | source leaprc.gaff
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| − | ###load TIP3P (water) force field
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| − | source leaprc.water.tip3p
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| − | ###load ions frcmod for the tip3p model
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| − | loadamberparams frcmod.ionsjc_tip3p
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| − | ###needed so we can use igb=8 model
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| − | set default PBradii mbondi3
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| − | ###load protein pdb file
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| − | rec=loadpdb ../001.structure/4s0v_protein_for_AMBER.pdb
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| − | #bond rec.649.SG rec.762.SG
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| − | #bond rec.454.SG rec.472.SG
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| − | #bond rec.444.SG rec.447.SG
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| − | #bond rec.328.SG rec.339.SG
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| − | #bond rec.385.SG rec.394.SG
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| − | ###load ligand frcmod/mol2
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| − | loadamberparams ../002.forceFieldParameters/4s0v_ligand.am1bcc.frcmod
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| − | lig=loadmol2 ../002.forceFieldParameters/4s0v_ligand_antechamber.mol2
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| − | ###create gase-phase complex
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| − | gascomplex= combine {rec lig}
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| − | ###write gas-phase pdb
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| − | savepdb gascomplex 4s0v.gas.complex.pdb
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| − | ###write gase-phase toplogy and coord files for MMGBSA calc
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| − | saveamberparm gascomplex 4s0v.complex.parm7 4s0v.gas.complex.rst7
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| − | saveamberparm rec 4s0v.gas.receptor.parm7 4s0v.gas.receptor.rst7
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| − | saveamberparm lig 4s0v.gas.ligand.parm7 4s0v.gas.ligand.rst7
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| − | ###create solvated complex (albeit redundant)
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| − | solvcomplex= combine {rec lig}
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| − | ###solvate the system
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| − | solvateoct solvcomplex TIP3PBOX 12.0
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| − | ###Neutralize system
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| − | addions solvcomplex Cl- 0
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| − | addions solvcomplex Na+ 0
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| − | #write solvated pdb file
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| − | savepdb solvcomplex 4s0v.wet.complex.pdb
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| − | ###check the system
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| − | charge solvcomplex
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| − | check solvcomplex
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| − | ###write solvated toplogy and coordinate file
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| − | saveamberparm solvcomplex 4s0v.wet.complex.parm7 4s0v.wet.complex.rst7
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| − | quit
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| − | We should again run this input file using a slurm script:
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| − | #!/bin/bash
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| − | #
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| − | #SBATCH --job-name=4s0v_tleap
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| − | #SBATCH --output=tleap_output.txt
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| − | #SBATCH --ntasks-per-node=24
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| − | #SBATCH --nodes=6
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| − | #SBATCH --time=48:00:00
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| − | #SBATCH -p long-24core
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| − | module load amber/16
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| − | tleap -f 4s0v_tleap.in
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| − | =Complex Minimization and Equilibration=
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