Optimized Sample Delivery for Time Resolved Serial Crystallography at X-ray Free Electron Lasers

Serial Femtosecond Crystallography (SFX) is a powerful X-ray diffraction technique that reveals ultrafast biomolecular dynamics at physiological temperatures. Delivering hydrated microcrystals into the intense X-ray beam is critical for all SFX experiments. As SFX advances, especially with time-resolved (TR) crystallography, innovations in sample delivery are essential to fully exploit modern light sources, enabling efficient and accurate studies on proteins of interest. TR experiments, which analyze reacting species along their reaction mechanism, are particularly demanding in terms of sample consumption. To address this, a 3D-printed Y-junction device was developed to introduce oil in either a co-flowing or droplet pattern acting as a sacrificial fluid, significantly reducing sample waste. The 3D-printed co-flow device, hybridized to a gas dynamic virtual nozzle (GDVN) and optimized for high-viscosity protein crystal delivery, reduced consumption by 95%, extended nozzle operation time, and enabled pump-probe TR-SFX with Photosystem II. Protein delivery for TR-SFX was further optimized by modifying surfaces within the 3D-printed structures to generate protein crystal laden-droplets segmented with oil. A modular, fully 3D-printed droplet injector enabled synchronized droplet delivery via electrical triggering and was successfully employed at the Linear Coherent Light Source. This approach, which fine-tuned droplet triggering parameters for synchronization with the X-ray Free Electron Laser (XFEL) pulses, significantly increased indexed patterns and reduced sample waste for both proteins, human NQO1 (NAD(P)H: quinone ii oxidoreductase 1) and phycocyanin. This uncovered the first SFX room-temperature structure of NQO1 at 2.5 Å resolution, revealing new insights into its catalytic function. Finally, droplet generation for mix-and-inject TR-SFX was demonstrated at the European XFEL (EuXFEL). This injection method synchronized droplets with the 10 Hz pulse trains of the EuXFEL and included a mixer to study NQO1 with nicotinamide adenine dinucleotide (NADH) at 305 ms and 1190 ms. The structure of NQO1 bound with NADH was resolved at 2.5 Å, conserving 97% of the sample compared to continuous injection. The interaction of NADH in the binding site of NQO1 provide new insights into its catalytic function and pave the way for further exploration of its reaction mechanism. These evolved sample delivery methods conserved up to 97% of sample during TR-SFX experiments.