The first step of the experiment that they did is to obtain a 3D model of PMP22 and RER1. The three-dimensional structure was determined by homology modeling using mouse PMP22 protein which shares 29% of its PMP22 genes with humans. The 3D model was then mutated at L16P and T118M to generate the mutant protein. RER1 was then docked to the constructed protein to simulate its behavior in the ER. The PMP22 system was then anchored to POPC bilayer membrane and submitted for .5ul MD simulations, which mimics what happens in real cell membranes.
The second step was the analysis that was done with the data that was obtained in the MD simulations (figure 1). The researchers did Principal component analysis, which reduces the complexity of the data and extracts the relevant motions of the atoms examined (figure 2, 6). In this analysis, eigenvectors, which describes the motion of the protein and eigenvalues, which in this case describes the total mobility associated with each eigenvector, were identified. The protein-protein docking calculations were also performed (figure 3,4,5). This calculation generates a detailed model that indicates where each atom is at any time. The effective binding energy was also obtained through the MMGBSA approach. This allows for the estimation of effective binding free energy, which is the energy needed to disassemble a protein or a molecule. The researchers then calculated the effective binding free energy for each of the PMP22- RER1, which is a measure of total free energy differences in the molecules that are involved (table 2,3). Clustering calculation was also performed to identify the most popular conformations.
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