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==IV. Simulation Analysis== | ==IV. Simulation Analysis== | ||
Revision as of 13:13, 6 April 2016
For additional Rizzo Lab tutorials see AMBER Tutorials.
In this tutorial, we will learn how to run a molecular dynamics simulation of a protein-ligand complex. We will then post-process that simulation by calculating structural fluctuations (with RMSD) and free energies of binding (MM-GBSA).
Contents
I. Introduction
II. Structural Preparation
Antechamber, Parmchk, tLeap
III. Simulation using pmemd
PMEMD
IV. Simulation Analysis
Ptraj
You should create another work directory for this step (004.ptraj, for example). 1. At first we want to concatenate the two 1ns trajectories together, stripping off the waters, and creating a .strip-file as an output. Below is the input file which will allow us to do so.
ptraj.1.in
trajin ../003.pmemd/10md.trj 1 1000 1 trajin ../003.pmemd/11md.trj 1 1000 1 trajout 4TKG.trj.strip nobox strip :WAT
The two sets of numbers 1 1000 1 give the input information about which frames are used for the Ptraj. The first two numbers 1 and 1000 specify the starting and ending snapshots from the trajectory file. The ending number of the snapshot doesn't need to be accurate because if you actually don't have enough snapshots in your trajectory file, Ptraj will read up to the last one you have. The last number 1 specifies the frequency of the snapshot saved, in this case, we are saving every frame of the trajectory file. And the last line of the input file will take away all the water molecules.
As the input file is prepared we can launch the first analysis as follows:
ptraj ../002.tleap/4TKG.com.wat.leap.parm ptraj.1.in > ptraj.1.log
As the output you will obtain 4TKG.trj.strip file which contains 2000 snapshots of the trajectory.
2. Later on we want to compare the output file just obtained to our reference file 4TKG.com.gas.leap.rst7, using the following input file.
ptraj.2.in
trajin 4TKG.trj.strip 1 2000 1 trajout 4TKG.com.trj.stripfit reference ../002.tleap/4TKG.com.gas.leap.rst7 rms reference out 4TKG.rmsd.CA.txt :1-206@CA
Since we have just concatenated the two trajectories, we will have 2000 snapshots in 4TKG.trj.strip. The third line in the input specifies the reference file, we have taken away all the water molecules during the first step, hence we are working here with the gas phase complex. The last line says we are calculating the rmsd for alpha carbon number 1 to 206.
And as we have this file filled out, we can run this step:
ptraj ../002.tleap/4TKG.com.gas.leap.parm ptraj.2.in > ptraj.2.log
The output file 4TKG.rmsd.CA.txt wil contain two columns, the first one is the number of frame and the second one stands for rmsd value.
3. Afterwards we will generate a similar file for ligand, using the following input file.
ptraj.3.in
trajin 4TKG.com.trj.stripfit 1 2000 1 reference ../002.tleap/4TKG.com.gas.leap.rst7 rms reference out 4TKG.lig.rmsd.txt :207@C*,N*,O*,S* nofit
And then run this step:
ptraj ../002.tleap/4TKG.com.gas.leap.parm ptraj.3.in > ptraj.3.log
By doing this we will compare the trajectory file to 4TKG.com.gas.leap.rst7 as well, but working with the ligand instead of the receptor (we specified that by pointing that we want to calculate the rmsd for carbon, nitrogen, oxygen and sulfur in residue 207.
The last two steps are to obtain energetic information about the system. To do this we take a trajectory file of the gas phase complex 4TKG.com.trj.stripfit, and want to create two more trajectory files containing the information on only receptor and only ligand correspondingly.
4. At this step we consider receptor only. The input file is provided below:
ptraj.4.in
trajin 4TKG.com.trj.stripfit 1 2000 1 trajout 4TKG.rec.trj.stripfit strip :207
And then run this step:
ptraj ../002.tleap/4TKG.com.gas.leap.parm ptraj.4.in > ptraj.4.log
5. And the same procedure for the ligand, with the following input file:
ptraj.5.in
trajin 4TKG.com.trj.stripfit 1 2000 1 trajout 4TKG.lig.trj.stripfit strip :1-206
And then run this step:
ptraj ../002.tleap/4TKG.com.gas.leap.parm ptraj.5.in > ptraj.5.log
6. (optional) Visualization
As we've gone through all these steps, the analysis is done. If you want to visualize the trajectories, you first need to copy the trajectory files to Herbie like this, for example (being a level above 004.PTRAJ directory):
scp 004.ptraj/ your_username@herbie.mathlab.sunysb.edu:~AMS536/amber_tutorial/004.ptraj
Now, launch VMD, then open one of the .prm7 files in 002.tleap. If you want to visualize the whole complex in gas state, you can open 4TKG.com.gas.leap.prm7 with AMBER7 Parm from 002.tleap and then 4TKG.com.trj.stripfit with AMBER coordinates from 004.ptraj. With these files, you can look at the real-time movement of the complex in the gas state. You can repeat this procedure to observe the real-time movement of the complex in the water state. Just open 4TKG.com.wat.leap.prm7 instead of 4TKG.com.gas.leap.prm7.
MM-GBSA Energy Calculation
Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) is a great method to calculate or estimate relative binding affinity of a ligand(s) to a receptor. The binding energy calculated from this method are also known as free energies of binding, where the more negative values indicate stronger binding. For this section, the topology files for the ligand, receptor and complex are needed.
Create a new directory:
mkdir 005.MMGBSA
Create an input file name
vim gb.rescore.in
Enter the following into the input file:
Single point GB energy calc &cntrl ntf = 1, ntb = 0, ntc = 2, idecomp= 0, igb = 5, saltcon= 0.00, gbsa = 2, surften= 1.0, offset = 0.09, extdiel= 78.5, cut = 99999.0, nsnb = 99999, imin = 5, maxcyc = 1, ncyc = 0, /
Create a tcsh/bash/csh script (run.sander.rescore.csh) with the following information:
#! /bin/tcsh #PBS -l nodes=1:ppn=1 #PBS -l walltime=48:00:00 #PBS -o zzz.qsub.out #PBS -e zzz.qsub.err #PBS -V #PBS -N mmgbsa set workdir = /nfs/user03/kbelfon/amber_tutorial/005.mmgbsa cd $workdir sander -O -i gb.rescore.in \ -o gb.rescore.out.com \ -p ../002.tleap/4TKG.com.gas.leap.prm7 \ -c ../002.tleap/4TKG.com.gas.leap.rst7 \ -y ../004.ptraj/4TKG.com.trj.stripfit \ -r restrt.com \ -ref ../002.tleap/4TKG.com.gas.leap.rst7 \ -x mdcrd.com \ -inf mdinfo.com sander -O -i gb.rescore.in \ -o gb.rescore.out.lig \ -p ../002.tleap/4TKG.lig.gas.leap.prm7 \ -c ../002.tleap/4TKG.lig.gas.leap.rst7 \ -y ../004.ptraj/4TKG.lig.trj.stripfit \ -r restrt.lig \ -ref ../002.tleap/4TKG.lig.gas.leap.rst7 \ -x mdcrd.lig \ -inf mdinfo.lig sander -O -i gb.rescore.in \ -o gb.rescore.out.test.rec \ -p ../002.tleap/4TKG.rec.gas.leap.prm7 \ -c ../002.tleap/4TKG.rec.gas.leap.rst7 \ -y ../004.ptraj/4TKG.rec.trj.stripfit \ -r restrt.rec \ -ref ../002.tleap/4TKG.rec.gas.leap.rst7 \ -x mdcrd.rec \ -inf mdinfo.rec exit
Execute this script on the seawulf cluster or machine(s) of your preference
qsub run.sander.rescore.csh
Three output files will be generated once the job is completed: gb.rescore.out.com, gb.rescore.out.lig, and gb.rescore.out.rec These files represent the single point energy calculation results for the complex (.com), the ligand (.lig) and the receptor (.rec). The energy will be output by the program Sander for each frame specified in the input file. The final results for one frame in one of the three files should look as the following:
FINAL RESULTS
NSTEP ENERGY RMS GMAX NAME NUMBER
1 5.9132E+03 2.0005E+01 1.2640E+02 C 159
BOND = 661.8980 ANGLE = 1751.7992 DIHED = 2581.7692
VDWAALS = -1696.6585 EEL = -13958.9335 EGB = -3125.9524
1-4 VDW = 747.0185 1-4 EEL = 7750.8118 RESTRAINT = 0.0000
ESURF = 11201.4791
minimization completed, ENE= 0.59132314E+04 RMS= 0.200047E+02
Extracting Data from MM-GBSA calculation and calculating Free energy of Binding From the output files above:
VDWAALS = ΔGvdw
EELS = ΔGcoul
EGB = ΔGpolar
SASA = ESURF
With this information ΔGnonpolar can be solved using equation(1):
ΔGnonpolar = SASA*0.00542 + 0.92 (1)
Once ΔGnonpolar is solved then ΔGmmgbsa can be determined by equation(2):
ΔGmmgbsa = ΔGvdw + ΔGcoul + ΔGpolar + ΔGnonpolar (2)
Solve equation 2 and 3 using the extracted information from all three output files. So therefore you should have ΔGmmgbsa for the complex, ligand and receptor
Finally ΔΔGbind can be calculated using equation (3):
ΔΔGbind = ΔGmmgbsa,complex – (ΔGmmgbsa,lig + ΔGmmgbsa,rec) (3)
Plot your ΔΔGbind and examine the plot for changes in the ligand position and the ΔΔGbind. Also, you should calculate the mean and standard deviation for your ΔΔGbind.
The following script (get.mmgbsa.sh) can be used to extract the energy from the three output files obtained above and calculate ΔΔGbind:
#! /bin/bash
# by Haoquan
echo com lig rec > namelist
LIST=`cat namelist`
for i in $LIST ; do
grep VDWAALS gb.rescore.out.$i | awk '{print $3}' > $i.vdw
grep EGB gb.rescore.out.$i | awk '{print $9}' > $i.polar
grep EELS gb.rescore.out.$i | awk '{print $6}' > $i.coul
grep ESURF gb.rescore.out.$i | awk '{print $3 * 0.00542 + 0.92}' > $i.surf
paste -d " " $i.vdw $i.polar $i.surf $i.coul | awk '{print $1 + $2 + $3 + $4}' > data.$i
rm $i.*
done
paste -d " " data.com data.lig data.rec | awk '{print $1 - $2 - $3}' > data.all
for ((j=1; j<=`wc -l data.all | awk '{print $1}'`; j+=1)) do
echo $j , >> time
done
paste -d " " time data.all > MMGBSA_vs_time.dat
rm namelist time data.*
Execute this script:
bash get.mmgbsa.sh
A text file called MMGBSA_vs_time.dat with x and y values separated by a space and comma should be created. Use XMGRACE to plot this dat file using the following command in Linux:
xmgrace MMGBSA_vs_time.dat
