Testing the limits: serial crystallography using unpatterned fixed targets.

Sheet-on-sheet (SOS) fixed-target chips are arguably the most versatile, cheapest and simplest sample-delivery method for ambient-temperature data acquisition using serial crystallography approaches at synchrotrons and X-ray free-electron lasers (XFELs). Their defining feature, the absence of any hard-patterned restrictions around crystals, is their strength as it removes limitations on crystal sizes or environments. However, it is also their weakness when it comes to limiting undesired effects on yet-to-be-irradiated crystals due to diffusing heat, radicals or gas originating from previous exposures. We explored whether SOS chips can be used for damage-free serial data collection on the new ID29 beamline at the ESRF-EBS, a fourth-generation synchrotron light source, as well as at the new Cristallina-MX station at SwissFEL. We collected serial data sets from microcrystals of the hemoprotein DtpAa, which was reported to have a highly radiation-sensitive iron-water bond length. The data sets differ in step size between exposures within and between lines of a serpentine-like data-acquisition scan. We observe no significant changes in the distance of the water ligand of the heme in the structures obtained from the ID29 SSX data. However, when compared with those collected at Cristallina-MX, the diffraction intensities collected at ID29 suggest global damage akin to Bragg termination occurring during the 90 µs exposure at ID29. Moreover, differences in the heme geometry and the proximal histidine-iron bond length point to local damage in all ID29 data sets regardless of the X-ray spacing. SFX data collected at Cristallina-MX show a phase transition of the DtpAa crystal lattice for X-ray step sizes of ≤20 µm. This phase transition might be caused by heating and/or hydrogen-gas-induced crystal dehydration. Vigilance remains required to safeguard against radiation damage at fourth-generation synchrotrons and XFELs.