Recent advances in quantifying protein conformational ensembles with dipolar EPR spectroscopy.

This perspective highlights recent applications and technological progress in dipolar electron paramagnetic resonance (EPR) spectroscopy, including double electron-electron resonance (DEER) spectroscopy. These methods provide nanoscale distance distributions between site-specific spin labels in biomacromolecules. The resulting data are particularly well suited for quantifying the structure and energetics of conformational ensembles of multi-state and flexible proteins. Recent applications span a wide range of systems and are accompanied by innovations in spin labeling, deuteration, in-cell measurements, integrative multi-technique approaches, and novel computational modeling methods combined with structure prediction tools.