PCA and aFEL
To identify the dominant motions in the nCOV-2019, SARS-COV, and all the mutants, PCA was performed. Most of the combined motions were captured by the first ten eigenvectors generated from the last 400 ns for SARS-COV, nCOV-2019, and extended mutant systems and the last 200 ns for other nCOV-2019 mutants. The percentage of the motions captured by the first three eigenvectors was 51% for nCOV-2019 and 68% for SARS-COV. In all mutations, more than 50% of the motions were captured by the first three eigenvectors. The first few PC’s describe the highest motions in a protein which are related to a functional motion such as binding or unbinding of protein from the receptor. The first three eigenvectors were used to calculate the aFEL using the last 400 ns of simulation for nCOV-2019 and SARS-COV as shown in Figure 5, which displays the variance in conformational motion. SARS-COV showed two distinct low free energy states shown as blue separated by a metastable state. There is a clear separation between the two regions by a free energy barrier of about 6–7.5 kcal/mol. These two states correspond to the loop motions in the L3 as well as the motion in C-terminal residues of SARS-COV. The L3 motion in nCOV-2019 is stabilized by the H-bond between N487 on RBD and Y83 on ACE2 as well as a π-stacking interaction between F486 and Y83. It is evident that the nCOV-2019 RBD is more stable than SASR-COV RBD and exists in one conformation whereas the SARS-COV interface fluctuates and the aFEL is separated into two different regions. The first two eigenvectors were used to calculate and plot the aFEL as a function of first two principal components using the last 200 ns of the simulation for mutant systems. aFEL for other systems is shown in Figure S4.
Figure 5  Mapping of the principal components of the RBD for the aFEL from the last 400 ns of simulations for SARS-COV (top row) and nCOV-2019-wt (bottom row). The color bar is relative to the lowest free energy state. In each system, the first eigenvector was used to construct the porcupine plots to visualize the most dominant movements (Figure S5). nCOV-2019 showed a small motion in L3 and the core region and the extended loop region are very rigid showing small cones in the porcupine plot. The core structure of the RBD remains dormant as the cones are blue in most of the regions (Figure S5-A). In SARS-COV, the C-terminal region shows large motions (Figure S5-B). Mutation N487A showed a large motion in L1 (Figure S5-C). Mutations Y449A, G447A, and E484A demonstrate large motions in L3 (Figure S5). Overall, these plots show the involvement of these residues in the dynamic stability of the RBD/ACE2 complex.