Integrative Molecular Dynamics Simulations Untangle Cross‐Linking Data to Unveil Mitochondrial Protein Distributions

Abstract Cross‐linking mass spectrometry (XL‐MS) enables the mapping of protein‐protein interactions on the cellular level. When applied to all compartments of mitochondria, the sheer number of cross‐links and connections can be overwhelming, rendering simple cluster analyses convoluted and uninformative. To address this limitation, we integrate the XL‐MS data, 3D electron microscopy data, and localization annotations with a supra coarse‐grained molecular dynamics simulation to sort all data, making clusters more accessible and interpretable. In the context of mitochondria, this method, through a total of 6.9 milliseconds of simulations, successfully identifies known, suggests unknown protein clusters, and reveals the distribution of inner mitochondrial membrane proteins allowing a more precise localization within compartments. Our integrative approach suggests, that two so‐far ambigiously placed proteins FAM162A and TMEM126A are localized in the cristae, which is validated through super resolution microscopy. Together, this demonstrates the strong potential of the presented approach.