Copyright © 2008 Washington University in St. Louis
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This tutorial assumes you've completed the APBS tutorial and are comfortable with PDB2PQR and APBS calculations for those applications. In order to perform the provided examples, you will need APBS 0.5.1 and quite a bit of computer time.
The hard part of writing this tutorial was done by Rob Yang. Rob was teaching himself MM-PBSA calculations with APBS by following the work of Massova and Kollman from:
It is strongly recommended that you read this paper before starting the lab! Rob's work involved substantial precursory simulations and other calculations (outlined below) which are beyond the scope of this lab. Therefore, the goal of this tutorial is simply to walk you through the steps required to perform MM-PBSA types of binding calculations and to provide the basic scripts for doing the accompanying computations.
![[Note]](images/note.png) | Note |
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This tutorial covers the original MM-PBSA methods for determining relative binding energies. However, this method has evolved quite a bit over the past 10 years and includes a number of modifications and generalizations. I'd be happy to talk about these changes with anyone interested in implicit solvent approaches for binding calculations. This tutorial outlines basic steps that should be common to most of these approaches. |
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This section outlines the preliminary calculations that Rob undertook to get the systems ready for MM-PBSA calculations. You do not need to redo these calculations; they are time-consuming and outside the scope of these labs/lectures.
One of the key ingredients for an MM-PBSA calculation is the molecular dynamics simulation(s) used to generate the initial conformations. Here are the steps Rob used to perform MD on the p53-mdm complex:
The PLOP program was used to add a missing residue (Q16) to the original PDB (1YCQ).
Keeping the crystallgraphic water, the PLOP structure was used as input to GROMACS; which was used to generate a truncated octahedron periodic solvent box with 14 Å extending in all directions from the protein.
The system was minimized in GROMACS with 200 steps of steepest decent minimization with complex fixed to allow water to move; 100000 steps of conjugated gradient with complex fixed; 100000 steps of conjugated gradient without complex fixed.
The system was gradually heated to 300K in 50K degree increments of 20ps NpT molecular dynamics runs at 1 atm.
The system was then subject to NpT molecular dynamics simulation at 300K and 1 atm for an additional 1.2 ns.
From the root-mean-squared deviation from the starting structure, the 300-1200 ps segment of the molecualr dynamics trajectory was used for further analysis. Frames were taken from this segment every 9 ps, for a total of 101 frames.
After the various conformations have been generated from the molecular dynamics run, the radii and charges need to be assigned for the Poisson-Boltzmann calculations. As discussed in the APBS tutorial, this assignment is generally handled through the PQR format. This section details the steps that Rob used to get PQR files for his complex.
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Note that this assignment assumes no titration state change upon formation of the complex. This can often be a bad assumption! |
The PARSE radii (from the PDB2PQR data file) were combined with the OPLS charges (from the GROMACS trajectory) to generate the PQR files with PDB2PQR.
Each frame represents the snapshot of the complex. 2 additional PQR files were extracted from this frame: the MDM2 portion (mon1) and the p53 portion (mon2). Rob did not run 3 separate MD's, everything's extracted from a single dynamics simulation of the complex.
Each frame was converted to a PQR file using PDB2PQR, with charges and radii as described above.
Mutants were made by mutating each frame using PLOP first, followed by PDB2PQR conversion to PQR format.
Rob's results are available for download here (160 MB tar.gz file). This archive includes the original PDB file as well as the file after PLOP modification.
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After downloading the lab archive, you will be working from the p53-mmpbsa/mmpbsa/ directory. All necessary scripts should be in the script/ subdirectory. Note that the mmpbsa directory contains data for a number of mutants as well as the wildtype complex. You probably won't have time to analyze all of these but it would be useful to try a mutant and wildtype. Ideally, you can work with your classmates to obtain the entire series of mutants.
We will begin the MM-PBSA process by calculating gas phase molecular mechanics energies for the complex and its components. You can accomplish this by running the script/mmgas.pl script. Since A single MD trajetory is used, the internal components (e.g., bonds, angles, etc.) of the energies should cancel between the complex and the components, leaving only the van der Waals and electrostatics terms. Invocation syntax for the script should be (something like):
$ ./script/mmpbsa.pl wt/input/pdb wt/input/misc path/to/your/output
| Note |
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You will likely need to modify |
As you know, MM-PBSA methods use surface areas to estimate[1] nonpolar solvation energies. You can determine surface areas (and therefore approximate nonpolar energies) through the script/sa.pl script. Invocation syntax should be:
$ ./script/sa.pl wt/input/pqr/mead_parse path/to/your/output script/apbs_apolar.in
| Note |
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You will likely need to modify |
We're now ready to perform a series of Poisson-Boltzmann electrostatics calculations for the polar part of the solvation energy. This is a very time-consuming step (8-9 hours on a single machine for a single mutant); please plan accordingly. The necessary APBS runs can be performed through the script/apbs_auto.pl script which can be invoked as
$ ./script/apbs_auto.pl wt/input/pqr/mead_parse path/to/your/output apbs_param/apbs_auto_good.in
Be sure to look at the apbs_param/apbs_auto_good.in APBS input file to assure that you understand the operations being performed.
| Note |
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You will likely need to modify |
APBS output contains lots of information and can be tedious to post-process. Run the script/apbs_process.pl script to automatically sum the results from the APBS calculations. This is invoked as
$ ./script/apbs_process.pl path/to/your/APBS-output
and will provide a summary of the APBS output as a *.sum file in the output directory.
After running the above steps, you should have several *.sum files in your molecular mechanics, surface area, and electrostatics output directories. These files summarize the information from these calculations and provide the data which should be combined to give your MM-PBSA approximation to the complex binding energy.
Due to the time-consuming nature of MM-PBSA calculations (e.g., the molecular dynamics simulation and the electrostatics computations), you are not obliged to do anything else with this lab. However, for your project, you may wish to use molecular dynamics results from earlier in the class to perform MM-PBSA binding calculations. The scripts and input files provided with this lab should provide a very good starting point for trying these types of calculations and eliminate some of the tedium associated with manually performing the various MM-PBSA calculations..