2023 AMBER tutorial 2 with PDBID 3WZE
Contents
Introduction
AMBER is employed to compute biomolecular simulations between a receptor and a ligand. AMBER allows us to look at changes in interaction over time. We will be using the protein and receptor, 3WZE.
Directory Setup
As always, we set up folders to keep us organized as we move generate files:
mkdir 001_structure mkdir 002_parameters mkdir 003_leap mkdir 004_equil mkdir 005_production
3WZE Structures
We will generate fresh mol2 and pdb files of both the receptor and the ligand.
Receptor
To avoid any unintended errors, we start with a fresh PDB file and generate the receptor in Chimera. First, open the PDB in Chimera. Then select the protein
Select -> Structure -> protein
Then
Select -> Invert (all models)
to select everything but the protein. Next we delete everything but the protein by
Actions -> Atoms/Bonds -> delete
Note: we do NOT add hydrogens or add charge. This will be addressed in TLEaP below. And now we save the file as BOTH a mol2 and a PDB (3wze_rec.mol2; 3wze_rec.pdb). Sometimes mol2 files are tricky to load in AMBER so it is best to have both just in case.
Ligand
To generate only the ligand, open 3wze.pdb and repeat:
Select -> Structure -> protein
This time we want to delete the protein so we delete the selection by
Actions -> Atoms/Bonds -> delete
Next we want to remove the detergent, acetone, and waters by
Select -> Residue -> HOH -> Actions -> Atoms/Bonds -> delete Select -> Residue -> DTT -> Actions -> Atoms/Bonds -> delete Select -> Residue -> ACT -> Actions -> Atoms/Bonds -> delete
We will once again add hydrogens to the ligand by selecting the ligand and then
Tools > Structure editing > Add H
And we will add charge by
Tools > Structure editing > Add Charge > (have Amber ff14SB and AM1-BCC selected) -> Ok
Now we save this as a mol2 file, 3wze_lig.mol2.
Go to the directory:
cd 001_structure
and transfer/save all files there.
Amber Simulation Parameters
Next, change directory to
cd 002_parameters
To generate parameters for the simulation, we must implement the following:
antechamber -i ../001_structure/3wze_lig_wH.mol2 -fi mol2 -o 3wze_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc 0
nc = 0 because the charge on the ligand is 0. If your ligand is non-zero, enter the appropriate charge at the end of this line. It may be helpful to check the protonation state of the ligand at pH 7. Once 3wze_ligand_antechamber.mol2 output file is generated, run parmch2:
parmchk2 -i 3wze_ligand_antechamber.mol2 -f mol2 -o 3wze_ligand.am1bcc.frcmod
TLEaP
Next we will generate the AMBER topology file and coordinate files. Switch to the directory:
cd 003_leap
Two types of files will be generated, parm7 (topology) and rst7 (coordinates). Create the input file:
vi leap.in
And then input the following (make sure you change the username):
#!/USERNAME/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 ../000_structure/3wze_rec.pdb
##@make disulfide bond
###load ligand frcmod/mol2
loadamberparams ../000_structure/3wze_ligand.am1bcc.frcmod
lig=loadmol2 ../000_structure/3wze_ligand_antechamber.mol2
###create gase-phase complex
gascomplex= combine {rec lig}
###write gas-phase pdb
savepdb gascomplex 3wze.gas.complex.pdb
###write gas-phase toplogy and coord files for MMGBSA calc
saveamberparm gascomplex 3wze.complex.parm7 3wze.gas.complex.rst7
saveamberparm rec 3wze.gas.receptor.parm7 3wze.gas.receptor.rst7
saveamberparm lig 3wze.gas.ligand.parm7 3wze.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 3wze.wet.complex.pdb
###check the system
charge solvcomplex
check solvcomplex
###write solvated toplogy and coordinate file
saveamberparm solvcomplex 3wze.wet.complex.parm7 3wze.wet.complex.rst7
quit
Once the files are generated, transfer the parm7 and rst7 files to the local environment. You can run them in Chimera to check the build. Open the protein, then open TOOLS--MD/ENSEMBLE ANALYSIS--MD MOVIE. Open the parm7 in prmtop box and then add the rst7 as a trajectory. Then click OK.
Equilibration
Next, we will minimize and equilibrate the system over nine successive steps. For information about the different parameters see the AMBER16 manual.
Change directory to:
cd 004_equil
The first step is to create the new file:
vi 01.min.md
And insert the following: 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 /
You'll want to pay close attention to the restraint mask - it lifts in later steps. Next:
vi 02.equil.mdin
Insert:
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 /
Then:
vi 03.min.mdin
and insert the following text:
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
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 /
Then:
vi 05.min.mdin
and insert the following text:
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
And:
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 /
Then:
vi 07.equil.mdin
Insert:
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
Here, you will need to specify the exact residues of your COMPLEX to enter at the restraintmask line. This means the total protein residues plus the ligand. Residue numbers which can be found in the wet.complex.pdb file generated in the 003_leap directory. So here, 3WZE is 298 residues plus the ligand is 299.
Insert the following: MD simulations &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=":1-299@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 /
Next:
vi 09.equil.mdin
Insert the following paying attention to the restraintmask line once more:
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=":1-299@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 /
All nine files will be run simultaneously. First we create the file:
vi mdequilibration.sh
And insert the script:
#!/bin/bash
#
#SBATCH --job-name=3wze_equilibration
#SBATCH --output=equilibration_output.txt
#SBATCH --ntasks-per-node=24
#SBATCH --nodes=1
#SBATCH --time=48:00:00
#SBATCH -p long-24core
module load amber/16
do_parallel="mpirun -np 80 pmemd.MPI"
prmtop="../003.tleap/3wze.wet.complex.prmtop"
coords="../003.tleap/3wze.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
$do_parallel -O -i ${input}.mdin -o ${input}.mdout -p $prmtop -c ${coords}.rst7 -ref ${coords}.rst7 -x ${input}.trj -inf ${input}.info -r ${input}.rst7
coords=$input
done
- Keep in mind that the 24 core is likely discontinued as of May 2023.
Submit to the queue:
sbatch mdequilibration.sh
Production
To generate the actual MD simulation, change to the following directory:
cd 005_production
And open the following file:
vi 10.prod.mdin
Note the restraint mask should include ONLY protein residues this time. Insert the following text:
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=":1-298@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 /
This will take a very long time to run on CPUs so here it may be advisable to run on the GPU. Create the file:
vi mdproduction.sh
And insert the following text:
!/bin/sh
#SBATCH --job-name=3wze_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/3wze.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` "
Submit to the queue.
sbatch mdproduction.sh
MD Analysis
Analysis and inspection of the RMSD, hydrogen bonds, and free energies of binding can now be evaluated.
RMSD
First, we create a file to remove the waters and undo changes from the production trajectory.
vi cpptraj_strip_wat.in
And insert the following text:
#!/usr/bin/sh parm ../002_leap/3wze.wet.complex.parm7 #read in trajectory trajin ../004_production/10.prod.trj #read in reference reference ../002_leap/3wze.wet.complex.rst7 #compute RMSD + align CA to crystal structure rmsd rms1 reference :1-299@CA #strip solvent strip :WAT:Na+:Cl- #create gas-phase trajectory trajout 3wze_stripfit_restrained_gas.trj nobox
Reference mask once again contains protein residues + ligand. Now, run the following command:
cpptraj -i cpptraj_strip_wat.in
Next, we create the file to generate the ligand RMSD:
vi cpptraj_rmsd_lig.in
And input the following text:
#!/usr/bin/sh #trajin the trajectory trajin 3wze_stripfit_restrained_gas.trj #read in reference reference ../003.tleap/3wze.gas.complex.rst7 #compute RMSD (do not fit internal geometris first, included rigid body motions, convert frames to ns (framenum*.005) rmsd rms1 ":299&!(@H=)" nofit mass out 3wze_lig_restrained_rmsd_nofit.dat time .005 #histogram the nofit rmsd histogram rms1,*,*,.1,* norm out 3wze_lig_restrained_rmsd_nofit_histogram.dat
Run the command:
cpptraj -p ../003.tleap/3wze.complex.parm7 -i cpp_rmsd_lig.in
Next, we will calculate RMSD of the receptor. Create the file:
vi cpptraj_rmsd_rec.in
And input the following:
#!/usr/bin/sh #trajin the trajectory trajin 3wze_stripfit_restrained_gas.trj #read in reference reference ../003.tleap/3wze.gas.complex.rst7 #compute RMSD (do not fit internal geometries first, included rigid body motions, convert frames to ns (framenum*.005) rmsd rms1 ":1-298&!(@H=)" nofit mass out 3wze_receptor_restrained_rmsd_nofit.dat time .005 #histogram the nofit rmsd histogram rms1,*,*,.1,* norm out 3wze_receptor_restrained_rmsd_nofit_histogram.dat
Restraint mask here only contains protein residues. Now run the command:
cpptraj -p ../003.tleap/3wze.complex.parm7 -i cpp_rmsd_receptor.in
Hydrogen Bonding
Next, we can calculate the number of hydrogen bonds. Create the file:
vi cpptraj_hbond.in
And insert the text:
!/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 :1-299 out 3wze_hbond.out avgout 3wze_hbond_avg.dat solventdonor :WAT solventacceptor :WAT@-O nointramol brid\ geout 3wze_bridge-water.dat dist 3.0 angle 140
Once again, include both protein and ligand residues (here, 299). Run the command:
cpptraj -p ../003.tleap/3wze.wet.complex.prmtop -i cpp_hbonds.in
MM-GBSA
And lastly, we calculate the free energy of binding. Create the file:
vi mmgbsa.in
And input the following text:
mmgbsa 3wze 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, /
This is a long computation and so we create a script:
vi mmgbsa.sh
With the text:
#bin/bash
#SBATCH --time=2-00:00:00
#SBATCH --nodes=2
#SBATCH --ntasks=28
#SBATCH --job-name=3wze_MMGBSA
#SBATCH --output=3wze_MMGBSA.log
#SBATCH -p extended-28core
#Define topology files
solv_parm="../003.tleap/3wze.wet.complex.prmtop"
complex_parm="../003.tleap/3wze.complex.parm7"
receptor_parm="../003.tleap/3wze.gas.receptor.parm7"
lig_parm="../003.tleap/3wze.gas.ligand.parm7"
trajectory="../005.production/10_prod.trj"
MMPBSA.py -O -i mmgbsa.in \
-o 3wze_mmgbsa_results.dat \
-eo 3wze_mmgbsa_perframe.dat \
-sp ${solv_parm} \
-cp ${complex_parm} \
-rp ${receptor_parm} \
-lp ${lig_parm} \
-y ${trajectory}
And execute with:
sbatch mmgbsa.sh
