Precision DEER Distances from Spin-Label Ensemble Refinement

Double electron-electron resonance (DEER) experiments probe nano-meter-scale distances in spin-labeled proteins and nucleic acids. Rotamer libraries of the covalently attached spin-labels help reduce position uncertainties. Here we show that rotamer reweighting is essential for precision distance measurements, making it possible to resolve Angstrom-scale domain motions. We analyze extensive DEER measurements on the three N-terminal polypeptide transport-associated (POTRA) domains of the outer membrane protein Omp85. Using the Bayesian inference of ensembles maximum-entropy method, we extract rotamer weights from the DEER measurements. Small weight changes suffice to eliminate otherwise significant discrepancies between experiments and model and unmask 1-3 angstrom domain motions relative to the crystal structure. Rotamer-weight refinement is a simple yet powerful tool for precision distance measurements that should be broadly applicable to label-based measurements including DEER, paramagnetic relaxation enhancement, and fluorescence resonance energy transfer (FRET).