Identification of distinct structural conformations of PPARγ from CRYO-EM maps using a shotgun molecular dynamics flexible fitting and normal mode analysis (MDFF-NM) approach

Peroxisome proliferator-activated receptors, also known as PPARs, are responsible for regulating gene expression and participating in activities related to inflammation, lipids, and glucose. In this work, we apply a shotgun molecular dynamics flexible fitting and normal mode analysis methodology (MDFF_NM) to explore the conformational landscape of peroxisome proliferator-activated receptor γ (PPARγ) based on information from the  cryo-electron microscopy (cryo-EM) density maps. MDFF_NM is a multi-replica strategy in which independent simulations sample diverse transition pathways from an initial to a target conformation by combining the restraining potential of MDFF with intrinsic harmonic motions derived from normal modes. This dual-constraint framework not only yields high-quality flexible fits into experimental densities but also generates a plausible ensemble of intermediate meta-states. We have implemented clustering of replica trajectories to identify predominant conformational basins and used the mean density of each cluster to refine fitting performance. Preliminary results demonstrate that (i) clustering increases convergence of flexible fitting and reduces overfitting in highly mobile regions, (ii) fitting to mean maps of top-ranking clusters produces atomic models with improved cross-validation scores, and (iii) combining meta-states with elevated Q-scores into consensus density maps enables de novo reconstruction of flexible loops that are otherwise unresolved. The success of MDFF_NM in recovering multiple accessible meta-states of PPARγ highlights its utility not only for structural refinement but also for mapping dynamic ensembles that underpin receptor function and ligand specificity. This approach promises to advance our understanding of allosteric regulation in nuclear receptors and to facilitate structure-based drug design targeting conformationally heterogeneous proteins.