## 1 Structural Biology at XFEL Sources
### Membrane Protein Serial Crystallography at MHz Rates  
Since membrane proteins generally have low expression levels, it is difficult to yield a large amount of protein for crystallization. Furthermore, crystallization often results in small crystals with large cell dimensions, and hence very few unit cells within a given crystal. For most traditional X-ray sources, a best-case scenario may still result in low-resolution data, as diffraction intensity is related, approximately, to the square of the number of unit cells. For example, photosystem I (PSI, or plastocyanin-ferredoxin oxidoreductase) belongs to a monoclinic crystal system with unit cell parameters of a = 279, b = 165, c = 284 A and \(\beta\) = 119.3\({}^{\circ}\)[56], and lysozyme belonging to a tetragonal crystal system with unit cell parameters of a = b = 79.2 and c = 37.7 A [57]. One would expect the lysozyme to provide stronger reflections, as the unit cell is approximately 46 times smaller in volume than the PSI. The N\({}^{2}\) rule of thumb would suggest the measured data to be 46\({}^{2}\), or approximately 2136 times weaker from the larger unit cell. [58]. Experiments by [PERSON] et al. successfully recorded diffraction images of crystallized cyanobacterial PSI at the EuXFEL using 5 \(\times\) 5 \(\times\) 15 \(\upmu\)m\({}^{3}\) crystals [56]. The collected data were comparable with previously studied synchrotron data measuring a cell dimension of 279, 165, 284 A, suggesting similar  
Figure 8: Data quality metrics, as determined from each pulse number, show that the structural data are independent of pulse number and that there is no systematic change in data quality across the train. Any section of the train can be utilized for data collection without compromise. Notably, the repetitive dips in data quality were due to known detector behavior and not due to the experiment itself. Figure originally published in Struct. Dyn. [52]; licensed under a Creative Commons Attribution (CC BY) license. crystallization and cell packing. Supplementary data provided by [PERSON] et al. stated that this was approximately 33 unit cells per crystal, while the lysozyme data studied by [PERSON] et al. were approximately 1400 unit cells. However, despite the significant decrease in the number of unit cells, [PERSON] et al. were able to determine the molecular structure to 2.9 A resolution. Data were collected with 30 pulses per train at 1.1 MHz using a sample injection speed of 50 m s\({}^{-1}\). They collected 7,719,186 images of PSI over a selection of detector distances to the sample (168, 233, and 327 mm). The 168 mm dataset was shown to contain significant overlap of Bragg reflections in the low-resolution shells. So-called _spot overlap_ creates difficulties in determining the local background and peak intensity. Extreme cases can result in multiple indistinguishable peaks measured as a single spot. Pixel size and unit cell size dictate how close a detector can be moved for a given sample and are therefore a limiting factor in maximum resolution of the data. As the detector distance is decreased, the solid angle across a given pixel as seen from the interaction region becomes greater, meaning any reflections measured within that solid angle will be integrated by a single pixel. Conversely, the solid angle is decreased as the detector-to-sample distance is increased, providing better peak separation as measured by the number of pixels. However, moving the detector further away from the sample also limits the total solid angle of the entire detector and therefore decreases the highest possible resolution. In conjunction with crystal quality and injection methods, consideration of detector position and pixel size is crucial to record the highest quality data possible. The successful indexing of the 233 and 327 mm sample-to-detector distance datasets was then compared to synchrotron data collected at ESRF [59]. By restricting the data to equal resolution, it was found that the EuXFEL data were similar and representative of the previous studies.