as the sum of the free energy differences between all pairs of
alchemical intermediate states [5]. Here, we will focus in particular
on absolute binding free energy (ABFE) calculations that make use
of such an alchemical cycle, in which the ligand is nonphysically
“removed” from the solution environment and “inserted” into the
protein’s binding site. Note that the termabsoluterefers to the fact
that a binding free energyΔGb, rather than a binding free energy
difference (ΔΔGb), is being calculated.
The advantage of the alchemical approach as compared to
computationally cheaper ones is that it is expected to be more
accurate due to its ability to return the exact binding free energy
for the physical model used [1, 2, 6]. As will become evident in this
chapter, however, this comes at a high computational cost. The
method is also in principle general, meaning it can be applied to any
protein–ligand pair, as long as the sampling challenges can be
addressed. Potential applications of the approach are the ranking
of ligands with different scaffolds binding to a specific protein, the
accurate rescoring of docking poses, and the prediction of ligand
selectivity [7, 8].
Here, we present a guide on how to carry out alchemical ABFE
calculations based on a standard protocol that uses equilibrium MD
simulations. In particular, we focus on the more practical aspects of
setting up the calculations alongside a brief overview of the theory
and technical details. Throughout the text, we direct the reader
who would like to further explore certain technical topics toward
more specialized reviews and chapters. Despite this being a guide
that aims to explain how to set up and run the calculations in simple
terms, at this point in time these calculations should still be consid-
ered an advanced approach. Consequently, we recommend that the
reader first familiarize themselves with ligand parametrization and
simulations of protein–ligand complexes. Some experience with
solvation free energy calculations may also be beneficial, as these
represent part of the thermodynamic cycle used for the binding free
energy calculations.
The chapter is organized as follows. First, we provide an over-
view of the theoretical aspects of binding free energy, including its
definition, how it can be extracted from computer simulations, and
the alchemical thermodynamic cycle we will use to calculate
it. Then, we touch upon the software and hardware requirements
for the calculations. Finally, we discuss the protocol step by step,
pointing out potential pitfalls and issues. In Subheading5, we show
how certain steps can be carried out using the Gromacs 2016
simulations package. This chapter is accompanied by a tutorial
(Absolute Binding Free Energy—Gromacs 2016) available on
http://www.alchemistry.org, where the reader can find the input files
needed to practice with an example calculation.200 Matteo Aldeghi et al.