During feature selection, only distances between C α atoms were used, which is the default setting for af2rave. With only 142 MSA sequences, reduced MSA AlphaFold2 generated structures with substantial diversity in the flexible regions. The backbone RMSD of these structures compared to the reference structure 6M0J reached up to 20 Å, whereas for the majority of the reference MD trajectory, it was within 5 Å(Figure ). However, our simulations showed that these structures, which deviated significantly from the experimental reference, could remain stable for the first 100 ns of simulation (Figure ). This suggests that the reduced MSA structures were too far removed from the conformational landscape explored by unbiased MD. Nevertheless, they may still represent relevant and potentially metastable states. Given this, we shifted from using reduced MSA AlphaFold2 to utilizing all available MSA information. This adjustment yielded structures that were more consistent with the MD data and more relevant to the system under study. In this test, the MSA depth, which governs the degree of structural diversity, ultimately depends on the user's decision and requires knowledge of the type of diversity being investigated.