Polymer fixed-targets for time-resolved serial protein crystallography at XFELs and synchrotrons

Serial crystallography at X-ray free electron lasers (XFELs) and synchrotron light sources, termed serial femto-second crystallography (SFX) and serial synchrotron crystallography (SSX) respectively, has become an effective method for determining macromolecular structures at room or near-physiological temperatures with minimal radiation damage. To support these experiments, various delivery methods have been developed, with fixed-targets based on micro-pattern solid-supports or chips proving to be particularly reliable. Fixed-targets reduce sample consumption, facilitate rapid optimization of sample loading, and are compatible with high-throughput technologies. The primary focus for this research was to create a novel polymer based fixed-target, the MIcro-Structured Polymer based fixed-target (MISP-chip), optimized for minimal background noise, cost-effective production, user-friendly handling, durability, high reusability, and applicability to both serial crystallography and pump-probe experiments. This fixed-target was aimed to be developed and established as a novel fixed-target delivery method for serial crystallography at synchrotrons and XFELs, specifically tailored for SwissFEL’s CristallinaMX experimental station. Using silicon microfabrication and polymer replication technologies, we have designed inverted pyramidal shaped wells in membranes ranging from 25-50 µm in thickness. This design enables crystals to funnel into predefined positions, optimizing the hit-rate of the probing X-ray beam. The polymer-based film provides low x-ray absorption and scattering background, high design flexibility and the potential for mass-fabrication at low cost. A clear COP and opaque COC chip were made for conducting standard serial and pump-probe experiments. Simultaneously, efforts were dedicated to investigating ligand binding interactions aiming to optimize a photocaged biotin-streptavidin system for pump-probe TR-SX experiments in attempt to reveal the structural dynamics of the binding events by using coumarins as the photocage.