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GMXPBSA 2.1: a GROMACS tool to perform MM/PBSA and computational alaninescanning C. Paissonia, D. Spiliotopoulosa,b, G. Muscoa, A. Spitaleria,c,*a Biomolecular NMR Unit, S. Raffaele Scientific Institute , via Olgettina 58, Milan 20132, Italyb 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. IntroductionMM/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 topost-process a series of representative snapshots from MD trajectories. MM/PBSA has beensuccessfully applied to compute the binding free energy of numerous protein–ligand interactions [2-5]. The method expresses the free energy of binding as the difference between the free energy of thecomplex and the free energy of the receptor plus the ligand (end-state method). This difference isaveraged over a number of trajectory snapshots [6]. Of note, the MM/PBSA approach allows for arapid estimation of the variation in the free energy of binding, with the caveat that generally it doesnot reproduce the absolute binding free energy values. Nevertheless, it usually exhibits goodcorrelations with experiments, thus representing a fair compromise between efficiency and efficacyfor the calculation and comparison of binding free energy variations. The theory underlyingMM/PBSA approach has been described previously [6]. Briefly, the binding free energy of a proteinmolecule 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:<G> = <EMM> + <Gsolv> – T<SMM> (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 theintramolecular 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 continuummedium with a low dielectric constant (ε=1) to a continuum medium with the dielectric constant ofwater (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 aprobe of radius 1.4 Å.Herein, we present an updated and revised version of the tool, GMXPBSA 2.1 (Fig. 1). We haveintroduced 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 betweendifferent ligands; handling of multiple protein-ligands MD simulations to allow comparisons (e.g. betweenwild-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 usage2.1 GMXPBSA 2.1 calculation workflowGMXPBSA 2.1 is a user-friendly suite of Bash/Perl scripts that efficiently streamlines the set upprocedure and the calculation of binding free energies for an ensemble of complex structuresgenerated by GROMACS MD engine. The program workflow, (Figures 1 and 2) consists of threedifferent sequential steps comprising:1. gmxpbsa0.sh:In this step, the tool exploits the gmxpbsa0.sh script to setup the system and to perform preliminarycalculations including:

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