The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.
© 2021 Hustedt et al.