*2.6. Molecular Dynamics Simulations*

The possible conformations for the CDR3 were investigated with molecular dynamics (MD) simulations, starting from representative of the six different clusters including all the best 18 energy-minimized structures from PONDEROSA C/S modeling. All simulations show an initial increase of the RMSD from the first structure of the specific NOE-restrained cluster, followed by rather stable equilibration at the value of about 2.5 Å (Figure 8a). During the simulation, most of the structures fluctuate about an average conformation with lower RMSD with respect to the initial structure, as witnessed by the much lower residue root mean square fluctuations (RMSFs) on the superimposed residues (Figure 8b). Large RMSF values are observed at loops and in the region 100–120 encompassing the CDR3. This is observed in most simulations, although in one of the simulations the region 50–70 is also showing large fluctuations.

**Figure 8.** (**a**) RMSD with respect to the lowest energy structure of the NOE−restrained clusters as a function of time of the six MD simulations that were carried out starting from the minimized representative structures from the six clusters of the NOE−restrained PONDEROSA C/S models of Nb23. Black trace = cluster 1 (11 members); red trace = cluster 2 (2 members); green trace = cluster 3 (2 members); blue trace = cluster 4 (1 member); orange trace = cluster 5 (1 member); pale brown trace = cluster 6 (1 member). (**b**) RMSF in the same six MD simulations as in panel (**a**), as a function of the residue number of Nb23. The color code of the traces is the same as in panel (**a**).

MD confirms the proximity of Arg50 and Tyr104 sidechains in all of the simulations originating from the different clusters of PONDEROSA C/S energy-minimized conformers, with a geometry of either cation–π or π–stacking interaction in the snapshots of the simulation concerning the NOE-restrained best cluster.

An interesting observation is that the simulations starting from different minimized conformers of the PONDEROSA C/S clusters sample different regions of the conformational space, as can be seen by comparing the average RMSD at each residue for the ensemble of MD snapshots from each pair of simulations and for the ensemble of the pooled snapshots. An example is provided in Figure S4 with the pooling (dashed curve) of two of the MD snapshot ensembles depicted in Figure 8B. The large increase in RMSD upon pooling the two ensembles is indicative of large differences in the conformations about which the two MD simulations are fluctuating (see Figure S4).
