database and hydrophobicity of the protein. The prepared receptor
was aligned and oriented to the proper position in the POPC lipid
bilayer (length: 88 A ̊ inx-axis and 91 A ̊ iny-axis) and the over-
lapping lipids and water molecules were removed. Removal of the
overlapping lipid molecules may result in a vacuum between the
receptor and lipid molecules. However, this problem could be
circumvented during the first stage of equilibrium, where the vac-
uum tends to disappear as the lipid molecules relax around the
protein. Subsequently, the receptor in the membrane system was
solvated with explicit water molecules and ionized with 150 mM
KCl salt condition.2.1.2 Molecular
Dynamics Simulation Steps
Before running MD simulation, preparation of the system, topol-
ogy and force field files are necessary to start the process. The
topology file contains all the data about the input structure and
connectivity of the atoms in the system. The force field file contains
data about the interactions existing between each of the atoms or a
group of atoms connected in the initial structure. After setup, the
systems for the simulation need to be relaxed in a controlled
manner. In our MD protocol, the whole system was energy-
minimized using conjugate-gradient method in the order of lipid
membranes, water molecules, and entire molecules. Subsequently,
gradual heating of the system from 0 to 300 K using a 0.1 K interval
at each step was performed. Generally, equilibration of the system is
often carried out in two stages: first, the system is simulated under
NVT ensemble, where the number of molecules, volume, and
temperature are kept constant, followed by NPT ensemble (i.e.,
to couple a barostat to the simulation and maintain a constant
pressure) to more closely resemble the experimental conditions.
In our protocol, equilibration was carried out for 50 ns under
NVT ensemble at 300 K followed by the production runs for
300 ns under NPT ensemble (T¼300 K;P¼1 atm).2.1.3 Quantitative Quality
Assurance
of the Simulation
Regarding the quality of the simulation, it is necessary to perform
additional checks to test for the convergence of thermodynamic
parameters, such as temperature, pressure, energy (potential and
kinetic energies of the system), volume, density, and root mean
square deviation (RMSD). If any of the thermodynamic parameters
has not converged sufficiently, it is necessary to extend the required
simulation steps (seeNote 2). Particularly, in the case of membrane
protein simulation, it is crucial to check the stability of both protein
and lipid bilayer. The widely used metrics for checking the stability
of the protein is calculating the RMSD of Cα-atoms with respect to
the initial structure. However, in the case of lipids, it is essential to
measure the mean surface area per lipid, which acts as a reliable
indicator for molecular packing and membrane fluidity, and make a
direct comparison with the available experimental data. Other460 Shaherin Basith et al.