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GMXPBSA 2.1: a GROMACS tool to perform MM/PBSA and computational alanine
scanning
C. Paissonia, D. Spiliotopoulosa,b, G. Muscoa, A. Spitaleria,c,*
a

Biomolecular NMR Unit, S. Raffaele Scientific Institute , via Olgettina 58, Milan 20132, Italy

b

Present address: Computational Structural Biology Biochemisches Institut Universität Zürich, Winterthurerstrasse

190, CH- 8057 Zürich, Switzerland.
c

Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy.

*

Corresponding author at: Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego, 30, Genoa

16163, Italy. E-mail address: andrea.spitaleri@iit.it

1. Introduction
MM/PBSA is a versatile method to calculate the binding free energies of a protein–ligand complex
[1]. It incorporates the effects of thermal averaging with a force field/continuum solvent model to
post-process a series of representative snapshots from MD trajectories. MM/PBSA has been
successfully applied to compute the binding free energy of numerous protein–ligand interactions [25]. The method expresses the free energy of binding as the difference between the free energy of the
complex and the free energy of the receptor plus the ligand (end-state method). This difference is
averaged over a number of trajectory snapshots [6]. Of note, the MM/PBSA approach allows for a
rapid estimation of the variation in the free energy of binding, with the caveat that generally it does
not reproduce the absolute binding free energy values. Nevertheless, it usually exhibits good
correlations with experiments, thus representing a fair compromise between efficiency and efficacy
for the calculation and comparison of binding free energy variations. The theory underlying
MM/PBSA approach has been described previously [6]. Briefly, the binding free energy of a protein
molecule to a ligand molecule in solution is defined as:
ΔGbinding = Gcomplex – (Gprotein + Gligand) (1)
A MD simulation is performed to generate a thermodynamically weighted ensemble of structures.
The free energy term is calculated as an average over the considered structures:
 =  +  – T (2)
The energetic term EMM is defined as:
EMM = Eint + Ecoul + ELJ (3)
where Eint indicates bond, angle, and torsional angle energies, and Ecoul and ELJ denote the
intramolecular electrostatic and Lennard-Jones energies, respectively.
The solvation term Gsolv in Eq. 4 is split into polar Gpolar and nonpolar contributions, Gnonpolar:
Gsolv = Gpolar + Gnonpolar (4)

GMXPBSA 2.1 calculates Gpolar and Gnonpolar with Adaptive Poisson-Boltzmann Solver (APBS)
program [7].
The polar contribution Gpolar refers to the energy required to transfer the solute from a continuum
medium with a low dielectric constant (ε=1) to a continuum medium with the dielectric constant of
water (ε=80). Gpolar is calculated using the non linearized or linearized Poisson Boltzmann equation.
The nonpolar contribution Gnonpolar is considered proportional to the solvent accessible surface area
(SASA):
Gnonpolar = γ SASA + β (5)
where γ = 0.0227 kJ mol–1 Å–2 and β = 0 kJ mol–1 [8]. The dielectric boundary is defined using a
probe of radius 1.4 Å.
Herein, we present an updated and revised version of the tool, GMXPBSA 2.1 (Fig. 1). We have
introduced in GMXPBSA 2.1 the following improvements with respect to the previous version [11]:
 control of the input and output options;
 automatic setup and a posteriori CAS calculations;
 CAS calculations on a single residues or on a set of residues simultaneously;
 handling of multiple protein-ligands MD simulations to allow comparisons between
different ligands;
 handling of multiple protein-ligands MD simulations to allow comparisons (e.g. between
wild-type complex and non-alanine mutants);
 handling of APBS calculations on a multi core system (distributed calculations in cluster).
 possibility to use custom van der Waals radii;
 check and restart of the failed MM/PBSA calculations;
 statistical analysis of the results.
2. Program usage
2.1 GMXPBSA 2.1 calculation workflow
GMXPBSA 2.1 is a user-friendly suite of Bash/Perl scripts that efficiently streamlines the set up
procedure and the calculation of binding free energies for an ensemble of complex structures
generated by GROMACS MD engine. The program workflow, (Figures 1 and 2) consists of three
different sequential steps comprising:
1. gmxpbsa0.sh:
In this step, the tool exploits the gmxpbsa0.sh script to setup the system and to perform preliminary
calculations including:



check of the required input files and directories;



extraction of the frames of the complex from the MD simulations, subsequently split in the
protein and the ligand components by the GROMACS tools;



calculation of the Coulomb energy contributions using either GROMACS tools or the
“coulomb” program available in the APBS suite, and Lennard-Jones term using
GROMACS.

If the computational alanine scanning (CAS) calculation is required, the script performs alanine
mutations on the defined residues on every single extracted frames removing the side chains atoms
of the target residues up to the beta C atom (CB atom) and then recalculating the Coulomb and the
Lennard-Jones energy contributions of the structure containing the alanine mutant. It also generates
the grid and the input to perform the APBS calculations for each frame of the simulation. The latter
task is critical, since deletion of artefacts in the MM/PBSA calculation requires an exact matching
of the grid setup between all the system components (complex, protein and ligand).
2. gmxpbsa1.sh:
In this step, the gmxpbsa1.sh script computes the solvation polar and nonpolar energy contributions
using APBS program. These calculations can be distributed on a cluster or on a multi core
workstation.
3. gmxpbsa2.sh:
In this last step, the gmxpbsa2.sh script combines for all the frames the single terms,  and
 respectively, in order to calculate the final binding free energy value. It also checks and tries
to fix errors and/or failures occurring in the preceding step 2 (APBS calculations). Statistical
analysis is also performed computing average and standard error (SE). The SE is calculated as
follows: SE = σ/√N, where σ is the standard deviation and N is the number of structures (MD
frames) used in the calculation. The average Coulomb and Lennard-Jones values, the polar and
nonpolar solvation terms are calculated along each trajectory. If a value differs from the average
more than two standard deviations it is considered as an outlier and the corresponding frame is
excluded from the final calculation. However, it is always possible to check for outlier frames, since
their reference-numbers are stored in the WARNING.dat file.
2.2 Installation and execution of the program
Once the source code of the program GMXPBSAtool.tar.gz has been downloaded the user should
perform the following steps:
1.

extract the source code in a user defined location, e.g.. /home/myprogram/, by typing tar

zxvf GMXPBSAtool.tar.gz; set the GMXPBSAHOME environment variable in bash: export
GMXPBSAHOME=/home/myprogram/GMXPBSAtool; change the /home/myprogram to
whatever directory is appropriate for your machine; verify write permissions in the directory
tree, and execute permissions for the gmxpbsa0.sh, gmxpbsa1.sh and gmxpbsa2.sh scripts.
$GMXPBSAHOME should be also added to the PATH.
2. In order to perform MM/PBSA calculations, the user has to run the tool by typing
$GMXPBSAHOME/

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