Characterizing Enzyme Reactions in Microcrystals for Effective Mix-and-Inject Experiments using X-ray Free-Electron Lasers

Mix-and-inject serial crystallography is an emerging technique that utilizes X-ray free-electron lasers (XFELs) and microcrystalline samples to capture atomically detailed snapshots of biomolecules as they function. Early experiments have yielded exciting results; however, there are limited options to characterize reactions in crystallo in advance of the beamtime. Complementary measurements are needed to identify the best conditions and timescales for observing structural intermediates. Here, we describe the interface of XFEL compatible mixing injectors with rapid freeze-quenching and X-band EPR spectroscopy, permitting characterization of reactions in crystals under the same conditions as an XFEL experiment. We demonstrate this technology by tracking the reaction of azide with microcrystalline myoglobin, using only a fraction of the sample required for a mix-and-inject experiment. This spectroscopic method enables optimization of sample and mixer conditions to maximize the populations of intermediate states, eliminating the guesswork of current mix-and-inject experiments.