Integrative structural biology of protein-RNA interactions in four dimensions

Protein-RNA complexes underlie essential cellular processes and understanding their functional mechanisms requires structural analysis. Yet, their inherent flexibility, multi-valency, and dynamics make them challenging targets for structural biology. Integrative approaches, combining nuclear magnetic resonance (NMR), small-angle scattering, cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), crosslinking mass spectrometry, single-molecule techniques, and artificial intelligence (AI)-based predictions, have enabled the characterization of increasingly complex ribonucleoprotein (RNP) assemblies, in vitro and in their cellular contexts. These strategies have begun to capture molecular architecture and dynamic behaviors across time and space, paving the way for 4D structural biology. Here, we review recent developments in integrative modeling of protein-RNA complexes, highlighting advances in in-cell, 4D and condensate structural biology, and discuss how these approaches shape our understanding of RNP assembly, regulation, and function in physiologically relevant environments.