2022 AMBER tutorial 1 with PDBID 6ME2
Contents
Introduction
AMBER is a program designed for computing biomolecular simulations. In this tutorial, AMBER will be used to simulate the dynamics between the ligand and receptor of the 6ME2 PDB file.
Directory Setup
For this tutorial, it is recommended to define the following directories to stay organized:
mkdir 001_structure mkdir 002_parameters mkdir 003_leap mkdir 004_equil mkdir 005_production
6ME2 Structure Files
If the 6ME2 DOCK tutorials were followed before this AMBER tutorial, the user has some experience generating and prepping receptor and ligand filed from the original 6ME2 PDB file. However, it is recommended that the user NOT use the files generated in the 6ME2 DOCK tutorials and to instead start from scratch here, as this allows the chance to catch mistakes that may have been made but not recognized while following the initial DOCK tutorials.
To begin this tutorial, search the Protein Data Bank (PDB) website using the code 6ME2, or use the Fetch... button on the home screen of UCSF Chimera for this four-letter code. All the following structures should be saved to the following directory:
cd 001_structure
Receptor File Generation
To generate a fresh copy of the receptor file, open the 6me2.pdb file containing the ligand-receptor complex. Go to
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. Go to
File -> Save Mol2...
and save as 6me2_rec.mol2. Close Chimera.
Ligand File Generation
To generate the ligand file, open the 6me2.pdb file again, and go to
Select -> Structure -> protein
then
Actions -> Atoms/Bonds -> delete
to delete the receptor. Then, go to
Select -> Residue -> HOH -> Actions -> Atoms/Bonds -> delete
Select -> Residue -> OLA -> Actions -> Atoms/Bonds -> delete
Select -> Residue -> PEG -> Actions -> Atoms/Bonds -> delete
to delete everything except for the JEV residue, which is the ligand of interest here.
Note: there are problems with the file containing the ligand as-is. In order to proceed properly, it is necessary to make some changes to this file. While the file is still open, go to
Tools > Structure Editing > Rotamers
and change the non-standard YCM residue to the most probable rotamers of CYS.
After this is completed, go to
Tools > Structure editing > Add H
to add hydrogens to the ligand. Then, go to
Tools > Structure editing > Add Charge > (have Amber ff14SB and AM1-BCC selected) -> Ok
and then go to
File -> Save Mol2...
and save as 6me2_lig_dockprep.mol2. Close Chimera.
Generating Simulation Parameters
In order to generate parameters for this tutorial, switch to the following directory:
cd 002_parameters
To generate the parameters, run the following command:
antechamber -i ../001_structure/6me2_lig_wH.mol2 -fi mol2 -o 6me2_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0
Note the -nc flag is set to 0 in the above line; this should correspond to the protonation state of the ligand. If a user is following this tutorial to simulate dynamics on a structure other than 6ME2, be sure to double check and change this value accordingly.
After the 6me2_ligand_antechamber.mol2 output file is generated, run the following command to run parmch2:
parmchk2 -i 6me2_ligand_antechamber.mol2 -f mol2 -o 6me2_ligand.am1bcc.frcmod
Using TLeap
Now, switch to the following directory:
cd 003_leap
Where files will be generated to simulate the ligand and receptor, such that further calculations involving both structures--such as binding affinities, free energies, and trajectories. Two types of files will be generated that can be used to set up the system: parameter (parm7) and restart (rst7) files. Start the process by creating the following input file:
vi leap.in
And put the following into the input file:
#!/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/6me2_fresh.pdb
###load ligand frcmod/mol2
loadamberparams ../002_parameters/6me2_ligand.am1bcc.frcmod
lig=loadmol2 ../002_parameters/1HW9_ligand_antechamber.mol2
###create gase-phase complex
gascomplex= combine {rec lig}
###write gas-phase pdb
savepdb gascomplex 6me2.gas.complex.pdb
###write gase-phase toplogy and coord files for MMGBSA calc
saveamberparm gascomplex 6me2.complex.parm7 6me2.gas.complex.rst7
saveamberparm rec 6me2.gas.receptor.parm7 6me2.gas.receptor.rst7
saveamberparm lig 6me2.gas.ligand.parm7 6me2.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 6me2.wet.complex.pdb
###check the system
charge solvcomplex
check solvcomplex
###write solvated toplogy and coordinate file
saveamberparm solvcomplex 6me2.wet.complex.parm7 6me2.wet.complex.rst7
quit
After this runs, copy the parm7 (parameter) and rst7 (coordinate) files to the local machine, where they can be opened in Chimera. To open in Chimera, go to
Tools -> MD/Ensemble Analysis -> MD Movie --> Choose appropriate parameter and coordinate files to load
Equilibrating the system
Now, move to the following directory:
cd 004_equil
In this process, energy minimization and equilibration will be performed on the system. There are 9 input files that need to be generated for this file, as follows:
vi 01.min.mdin
With the following input:
Minimize all the hydrogens &cntrl imin=1, ! Minimize the initial structure ntmin=2, ! Use steepest descent Ryota Added 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="!@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 /
Next,
vi 02.equil.mdin
with input
MD simulation &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=":!@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 /
Next,
vi 03.min.mdin
with input
Minimize 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="!@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 /
Next,
vi 04.min.mdin
with input
Minimize 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="!@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 /
Next,
vi 05.min.mdin
with input
Minimize 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="!@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 /
Next,
vi 06.equil.mdin
with input
MD simulation &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="!@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 /
Next,
vi 07.equil.mdin
with input
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="!@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 /
Next,
vi 08.equil.mdin *See below for specific comments on this input
with input
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=":1-433@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 /
And finally,
vi 09.equil.mdin *See below for comments on this input
with input
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=":1-433@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 that the restraint mask in files 8 and 9 have to be specified to the relevant system being simulated. These are residue numbers which can be found in the wet.complex.pdb file generated in the 003_leap directory.
To submit all 9 input files as a job on Seawulf, create the following script:
vi mdequilibration.sh
with input
#!/bin/sh
#SBATCH --job-name=6me2_equilibration
#SBATCH --ntasks-per-node=40
#SBATCH --nodes=2
#SBATCH --time=8:00:00
#SBATCH -p long-40core
echo "started Equilibration on 'date' "
parm7="../003_leap/6me2.wet.complex.parm7"
coords="../003_leap/6me2.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
pmemd -O -i ${input}.mdin -o ${input}.mdout -p $parm7 -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r
${input}.rst7
coords=$input
done
echo " Finished equilibration on 'date' "
And submit this to the queue:
sbatch mdequilibration.sh
MD Production
Now, enter the following directory:
cd 005_production
and create the following production input file:
vi 10.prod.mdin
with inputs
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=":1-432@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 /
The restraint mask should be modified on this file as well to only include the receptor, not the ligand (i.e., it should be one fewer than the restraint mask value in the 8th input file from the previous directory).
Create a file that will be submitted to Seawulf:
vi mdproduction.sh
with the following:
!/bin/sh
#SBATCH --job-name=6me2_prod
#SBATCH --ntasks-per-node=24
#SBATCH --nodes=2
#SBATCH --time=7-00:00:00
#SBATCH -p extended-24core
echo "started Equilibration on 'date' "
parm7="../003_leap/6me2.wet.complex.parm7"
coords="../004_equil/09.equil"
MDINPUTS=( 10.prod)
for input in ${MDINPUTS[@]}; do
pmemd -O -i ${input}.mdin -o ${input}.mdout -p ${parm7} -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r
${input}.rst7
coords=$input
done
echo "Finished Equilibration on `date` "
And now submit this job to the queue:
sbatch mdproduction.sh
And note that this job will likely take a very long time, perhaps days.
MD Analysis
Since the trajectories have been generated, they can now be inspected using RMSD calculations, hydrogen bond analysis, and MMGBSA calculations.
RMSD Calculation
To perform the RMSD calculations, first create an input file which will delete the water and charges from the production trajectory.
vi cpptraj_strip_wat.in
with the following inputs
#!/usr/bin/sh parm ../003_leap/6me2.wet.complex.parm7 #read in trajectory trajin ../004_production/10.prod.trj #read in reference reference ../003_leap/6me2.wet.complex.rst7 #compute RMSD + align CA to crystal structure rmsd rms1 reference :1-263@CA #strip solvent strip :WAT:Na+:Cl- #create gas-phase trajectory trajout 6me2_stripfit_restrained_gas.trj nobox
The reference mask should contain both the ligand and receptor in its value.
Now run this file using the following command:
cpptraj -i cpptraj_strip_wat.in
Next, create another input file which will be used to calculate the RMSD of the ligand by typing
vi cpptraj_rmsd_lig.in
with inputs
#!/usr/bin/sh #trajin the trajectory trajin 6me2_stripfit_restrained_gas.trj #read in reference reference ../003_leap/6me2.gas.complex.rst7 #compute RMSD (do not fit internal geometris first, included rigid body motions, convert frames to ns (framenum*.005) rmsd rms1 ":254&!(@H=)" nofit mass out 6me2_lig_restrained_rmsd_nofit.dat time .005 #histogram the nofit rmsd histogram rms1,*,*,.1,* norm out 6me2_lig_restrained_rmsd_nofit_histogram.dat
And check that the reference mask only factors in the ligand in this file. Run this input file using the following command:
cpptraj -p ../003_leap/6me2.complex.parm7 -i cpptraj_rmsd_lig.in
Next, the receptor RMSD can be calculated by creating the following input file:
vi cpptraj_rmsd_rec.in
with inputs
#!/usr/bin/sh #trajin the trajectory trajin 6me2_stripfit_restrained_gas.trj #read in reference reference ../003_leap/6me2.gas.complex.rst7 #compute RMSD (do not fit internal geometries first, included rigid body motions, convert frames to ns (framenum*.005) rmsd rms1 ":1-253&!(@H=)" nofit mass out 6me2_rec_restrained_rmsd_nofit.dat time .005 #histogram the nofit rmsd histogram rms1,*,*,.1,* norm out 6me2_rec_restrained_rmsd_nofit_histogram.dat
And check that the reference mask only takes into account the receptor. Now run this file using the following command:
cpptraj -p ../003_leap/6me2.gas.complex.parm7 -i cpptraj_rmsd_rec.in
Calculating Number of Hydrogen Bonds
Next, the number of hydrogen bonds will be analyzed to see if any exist between the receptor and ligand. Create the following input file:
vi cpptraj_hbond.in
with inputs
!/usr/bin/sh #read in trajectory trajin ../004_production/10_prod.trj #wrap everything in one periodic cell - could cause problems, may comment out #autoimage if problems later autoimage #compute intra + water-mediated H-bonds hbond hb1 :1-263 out 6me2_calcipotriol_hbond.out avgout 6me2_calcipotriol_hbond_avg.dat solventdonor :WAT solventacceptor :WAT@-O nointramol brid\ geout 6me2_calcipotriol_bridge-water.dat dist 3.0 angle 140
Be sure that the hbobnd hb1 parameter corresponds to the residue numbers of the relevant ligand and receptor. Now, ru this input file with the following command:
cpptraj -p ../003_leap/6me2_wetcomplex.parm7 -i cpptraj_hbond.in
MMGBSA Calculation
Next, MMGBSA will be used to calculate free energies of binding in this system. First, create the following input file:
vi mmgbsa.in
with inputs
mmgbsa 6me2 analysis &general interval=1, netcdf=1, keep_files=0, / &gb igb=8 saltcon=0.0, surften=0.0072, surfoff=0.0, molsurf=0, / &nmode drms=0.001, maxcyc=10000, nminterval=250, nmendframe=2000, nmode_igb=1, /
To run this on Seawulf, create the following script:
vi mmgbsa.sh
and input the following
#bin/bash
#SBATCH --time=2-00:00:00
#SBATCH --nodes=2
#SBATCH --ntasks=28
#SBATCH --job-name=6me2_MMGBSA
#SBATCH --output=6me2_MMGBSA.log
#SBATCH -p extended-28core
#Define topology files
solv_parm="../003_leap/6me2.wet.complex.parm7"
complex_parm="../003_leap/6me2.complex.parm7"
receptor_parm="../003_leap/6me2.gas.receptor.parm7"
lig_parm="../003_leap/6me2.gas.ligand.parm7"
trajectory="10_prod.trj"
MMPBSA.py -O -i mmgbsa.in \
-o 6me2_mmgbsa_results.dat \
-eo 6me2_mmgbsa_perframe.dat \
-sp ${solv_parm} \
-cp ${complex_parm} \
-rp ${receptor_parm} \
-lp ${lig_parm} \
-y ${trajectory}
And finally, submit this as a job to the queue by typing
sbatch mmgbsa.sh
