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This perspective review describes emerging techniques and future opportunities for timeresolved serial femtosecond crystallography (TR‐SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS‐II will be available in the near future. One efficient use of these facilities for TR‐SFX relies on pump–probe experiments using a laser to trigger a reaction of light‐responsive proteins or mix‐and‐inject experiments for light‐unresponsive proteins. With the view to widen the application of TR‐SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light‐responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition ratecompatible sample delivery methods.

The Mn4CaO5 cluster site in the oxygen-evolving complex (OEC) of photosystem II (PSII) undergoes structural perturbations, such as those induced by Ca2+/Sr2+ exchanges or Ca/Mn removal. These changes have been known to induce long-range positive shifts (between +30 and +150 mV) in the redox potential of the primary quinone electron acceptor plastoquinone A (QA), which is located 40 Å from the OEC. To further investigate these effects, we reanalyzed the crystal structure of Sr-PSII resolved at 2.1 Å and compared it with the native Ca-PSII resolved at 1.9 Å. Here, we focus on the acceptor site and report the possible long-range interactions between the donor, Mn4Ca(Sr)O5 cluster, and acceptor sites.