2021 AMBER tutorial 1 with PDBID 1HW9
Contents
Introduction
In this tutorial, we'll be exploring the wonders of molecular dynamics through the use of AMBER 16. AMBER 16 we'll help study the binding characteristics of the ligand to the target protein!
Directory Setup
It's important to stay organized during this process as we'll be generating a number of files during the process. Overall, we'll be working within five directories. They should be created as follows:
mkdir 001_structure mkdir 002_parameters mkdir 003_leap mkdir 004_equil mkdir 005_production
Before proceeding, double-check that all directories have correctly been made.
1HW9 Structures
Let's move into our directory titled 001_structure.
cd 001_structure
Receptor
We need to retrieve a fresh copy of the receptor before proceeding forward. Even if you've done the DOCK tutorial, you should still retrieve a fresh .pdb just in case Chimera did something wrong during the preparation process. In order to do so, head over to the PDB website and type in 1HW9 into the header. It should take you straight to the webpage. Download the system as a pdb and open it in Chimera. Our focus will be on Chain A of the complex. In order to visualize the chain:
Select -> Chain -> A
Notice, however, that this does not remove Chains B, C, and D from our view. We need to perform this action manually.
Select -> Invert (Selected Molecules) -> Actions -> Atoms/Bonds -> Delete
After deletion, the only thing remaining should be Chain A of the original file. It is within your best interest to save this session as you'll need to come back to this file when preparing the ligand. Doing so will prevent you from having to do the above deletion step again.
File -> Save Session As ->
Now that the session has been saved, we can go ahead and isolate the receptor. While we've isolated Chain A, you'll notice that Simvastatin is still located on the molecule. In order to remove everything besides HMGR, we need to do the following:
Select -> Residue -> ADP -> Actions -> Atoms/Bonds -> Delete Select -> Residue -> HOH -> Actions -> Atoms/Bonds -> Delete Select -> Residue -> SIM -> Actions -> Atoms/Bonds -> Delete
With the receptor isolated, go ahead and save the file as
1HW9_fresh.pdb
Ligand
Open the session in Chimera we previously saved that consisted of Chain A with both the receptor and Simvastatin ligand. This time around, we'll be using the session file to isolate the ligand instead of the receptor.
Select -> Residue -> SIM -> Select -> Invert (Selected Molecules) -> Actions -> Atoms/Bonds -> Delete
We'll need to properly protonate the ligand in order for the AMBER calculations to function:
Tools -> Structure Editing -> Add H Tools -> Structure Editing -> Add Charge -> (have Amber ff14SB and AM1-BCC selected) -> Ok
Once done, make sure to save your file as:
1HW9_lig_wH.mol2
NOTE: Before proceeding, make sure both the ligand and receptor file you just created have been copied from your local computer to the 001_structure directory in your Seawulf account.
Simulation Parameters
Let's move into the second directory:
cd 002_parameters
Here, we'll be generating force field parameters for our ligand so that AMBER can use them in later calculations. We'll be doing this using antechamber and parmchk2:
antechamber -i ../001_structure/1HW9_lig_wH.mol2 -fi mol2 -o 1HW9_ligand_antechamber.mol2 -fo mol2 -at gaff2 -c bcc -rn LIG -nc -1
Notice the -nc flag at the end of the command line. Make sure that this value corresponds to the protonation state of your ligand! Once the output file is generated, we need to run parmch2 to generate a frcmod file:
parmchk2 -i 1HW9_ligand_antechamber.mol2 -f mol2 -o 1HW9_ligand.am1bcc.frcmod
TLeap System
Let's now move into the third directory:
cd 003_leap
In this stage, we'll be creating files to simulate the ligand and receptor as one system. This way, calculations like binding affinities and energies can be performed on the system as a whole. Fortunately, tleap will do this for us by generating parameter (parm7) and restart (rst7) files. In order to get started, we'll need to create the input file.
vi leap.in
Copy the following code into your new 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/1HW9_fresh.pdb
###load ligand frcmod/mol2
loadamberparams ../002_parameters/1HW9_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 1HW9.gas.complex.pdb
###write gase-phase toplogy and coord files for MMGBSA calc
saveamberparm gascomplex 1HW9.complex.parm7 1HW9.gas.complex.rst7
saveamberparm rec 1HW9.gas.receptor.parm7 1HW9.gas.receptor.rst7
saveamberparm lig 1HW9.gas.ligand.parm7 1HW9.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 1HW9.wet.complex.pdb
###check the system
charge solvcomplex
check solvcomplex
###write solvated toplogy and coordinate file
saveamberparm solvcomplex 1HW9.wet.complex.parm7 1HW9.wet.complex.rst7
quit
In order to now execute:
tleap -f leap.in
Once this is done, you should copy your parameter and coordinate files to your local computer and view them in Chimera. You want to make sure that everything looks correct before proceeding forward. For example, your protein ligand complex should not be outside the water molecule box that you just created. It's little things like this that can create headaches later on. In order to view in Chimera:
Tools -> MD/Ensemble Analysis -> MD Movie --> Choose appropriate parameter and coordinate files to load
Equilibration
Let's now move into our fourth directory:
cd 004_equil
We now need to perform energy minimization and equilibration similarly to how it was performed in the DOCK tutorial. Unfortunately, this also means we need to create 9 input files as this is a 9 step procedure: You're input files should be named and contain the following parameters:
vi 01.min.mdin
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 /
vi 02.equil.mdin
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 /
vi 03.min.mdin
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 /
vi 04.min.mdin
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 /
vi 05.min.mdin
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 /
vi 06.equil.mdin
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 /
vi 07.equil.mdin
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 /
vi 08.equil.mdin *See below for specific comments on this 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 /
vi 09.equil.mdin *See below for comments on this 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: The restraint mask lines in files 8 and 9 have to be specific to your system. These numbers correspond to residues in your system. These residue numbers can be found in the wet.complex.pdb file we generated back in step 3. File 8's restraint mask should include both the receptor and the ligand. You'll know the residue number of the ligand as the pdb will have is labeled as LIG. File 9's restraint mask is only the receptor so the value should actually be 1 less.
