## 1 Structural Biology at XFEL Sources
### Time-Resolved Serial Crystallography at MHz Rates  
We have seen from the previous examples that the SPB/SFX instrument at the EuXFEL enables a much faster throughput approach to XFEL structural biology than previously available. This vastly improved data collection rate is of extreme benefit for investigating the dynamics of biologically relevant systems, which requires the same amount of data as a static structure scaled by the number of time points of interest. Put simply, 10 frames of motion would require 10 times more data than a static structure of equal quality. The early user experiments at the EuXFEL have been invaluable in demonstrating the capabilities of SFX experiments at MHz rates. MHz SFX lends itself to more data collection in less time and therefore also better statistical analysis of difficult datasets than previously possible at lower-repetition-rate SFX facilities. For this reason, MHz SFX encourages novel experiments. Pump-probe experiments can investigate samples that have short-lived, optically-excited states or transient states while undergoing conformational change. Shortly before the X-ray pulse probes a sample, an additional external light source is transported to the interaction region to \"pump\" the sample into an excited or transient state. The time between pump and probe can be labelled as \(\Delta t\). Pump-probe experiments are most easily demonstrated with the use of an optical laser (the \"pump\"), where that laser interacts with an optically sensitive protein, such as PSI or PSII (Photosystem II, or water-plastoquinone oxidoreductase), before being probed by the X-ray pulse. Light-sensitive proteins are thought to go through many conformational changes before relaxing into a stable state. Observing and determining these conformational changes elucidates never-before-seen interactions, opening the possibility for future applications. Pump-probe experiments have been successful at LCLS [62] and other FELs [63]; however, with the newly available MHz source at EuXFEL, SPB/SFX is now opening up new frontiers into more rapid data collection of transient protein structures. As early as September 2019, [PERSON] et al. published successful femtosecond time-resolved SFX data collected at the SPB/SFX instrument at a MHz rate [64]. The photocycle of photoactive yellow protein (PYP) contained previously unknown transient states. PYP is a bacterial photosensor that undergoes a conformational photocycle consisting of many intermediates. The changes between  
Figure 9: Zoomed lysozyme diffraction pattern recorded on the AGIPD-1M detector. Gray-scale pixel intensity is measured in the “high” gain stage. Pixels colored red have been measured in the “medium” gain stage. Figure modified from original publication in Nat. Commun. [56]. states occur across a large range of temporal resolutions, from picoseconds to milliseconds. Structural information exists for most of the states within the photocycle, though spectroscopy data suggest a novel confirmation in the range of 1 to 100 picoseconds after entering the excitation state \(\mathrm{PG^{*}}\), Figure 10.  
PYP has been of significant interest due to its potential use in optically controlled biological reactions. Previously, [PERSON] et al. were able to determine the 3 ps time point at LCLS and the 100 ps time point at APS during separate experiments. Another experiment, conducted at SPB/SFX, injected PYP at 30 m s\({}^{-1}\) and matched with a 0.5 MHz intra-train rate, as supported by [PERSON] et al. [50]. The final experimental setup illuminated the sample 10 ps before the arrival of the second pulse. Each X-ray pulse was separated by 1.78 us, and each laser pulse was separated by 7.1 us. The resulting data collection returned four different time points as measured from the initial illumination (10 ps and 1.78, 3.56, and 5.33 us). Shown in Figure 11, the laser illumination overlapped with the second, third, and forth pulses and kept the first pulse dark, or not illuminated, then repeated this pattern across the entire pulse train.