The respective values in the presence and absence of LZE are -3.04 ± 0.39 kcal/mol and -3.32 ± 0.23 which are statistically the same. However, the important thing to note here is that the respective spreads (difference between extremes) in ∆G values for these systems are 1.54 kcal/mol (ranging from -3.97 to -2.43 kcal/mol) and 2.64 kcal/mol (ranging from -4.56 to -1.92 kcal/mol) which are both much larger than the difference between their mean ∆G values. This indicates the importance of performing ensembles in order to obtain statistically robust and reliable conclusions. For instance, taking the opposite extremes of ∆G direct values for both systems, we could have obtained differences of either -2.05 kcal/mol or 2.13 kcal/mol in the presence and absence of LZE, respectively, leading to diametrically opposite conclusions on its effect on the binding of RQN. But on performing ensemble simulations we are able to state with confidence that no statistically significant effect has been observed. Another important effect that has been observed is the emergence of a new binding site for RQN (denoted as C2), very close to the experimentally observed binding site (denoted as C), when binding to 3CLPro active site in the presence of LZE. Experimentally, it has been shown that the binding of LZE at allosteric site II displaces the loop 153-155 such that C α atom of TYR154 moves 2.8 Å, accompanied by a conformational change of ASP153. This loop is connected to loop 167-172 through a β-sheet strand 156-166 which is expected to cause a shift in the former as well. We have quantified this effect using our simulations. 5 replicas, wherein LZE binds to site B1, were processed to extract only the frames in which LZE is indeed bound to the said binding site and RMSDs calculated for loops 153-155 and 167-172 at each frame. Figure displays the time series of both these RMSD values for all replicas. It can be clearly seen that the RMSDs of both loops are correlated with both increasing/decreasing at the same time. This provides evidence for the displacement of loop 167-172 through LZE binding. Figure displays binding sites C (red) and C2 (blue) for RQN in the form of observed volume occupancy maps. Binding sites C and C2 have loops 167-172 and 186-191 in common (shown in green). Therefore, the binding of LZE at allosteric site II brings about conformational changes to the active site and creates enough space to let RQN bind at a slightly different location, very close to the original site. It appears that such a change does not have any substantial impact on the binding interactions of RQN with the residues of site C, thereby not affecting its binding affinity. However, its sampling frequency is certainly affected such that, in the absence of LZE, it is sampled by 10 out of 12 "long" replicas, whereas in its presence, it is sampled only by 3 out of 12 "long" replicas. On the other hand, site C2 is sampled in 2 out of 12 "long" replicas (exclusively in the presence of LZE). A similar effect has been observed in case of LZE with RQN present such that a new close-by binding site (denoted as B2) is sampled along with the crystallographically determined binding site (denoted as B1). Table and Figure include both such binding sites (for both RQN and LZE in presence of each other) as experimental binding sites and display results accordingly.