PROJECT SUMMARY/ABSTRACT Next generation structural biology experiments look to move beyond static observations of structure to a dynamic, time-resolved understanding of function. These next-level experiments are enabled by serial crystallography at X-ray free-electron lasers (XFELs) and microfocus synchrotron beamlines but are limited by issues of sample consumption. The long-term goal of this project is to democratize studies of protein structural dynamics by developing robust fixed-target mounting strategies for light-, chemical-, and electrically-triggered time-resolved protein crystallography experiments while maximizing sample utilization. This effort leverages the expertise of the SLAC National Accelerator Laboratory and the BioCARS facility at the Advanced Photon Source for testing and validation of new mounting strategies. These relationships will also enable rapid translation of developed technologies to the larger structural biology community. We have four initial aims to do this: Aim 1: Develop fixed-target strategies to facilitate the study of macromolecular crystallography targets while maintaining biological activity. We will design and fabricate X-ray compatible sample holders that maintain the stability of protein crystals over time while also allowing for easy sample loading, robotic handling, and in situ spectroscopy. A particular focus will include enabling data collection in a fully anaerobic environment, and the technologies developed here will serve as a basis for efforts in subsequent Aims. Aim 2: Develop fixed-target platforms for photo-triggering of reactions for analysis via time-resolved serial crystallography. We will develop X-ray compatible sample holders that take advantage of hydrodynamic forces to create large-scale arrays of crystals for use in serial crystallography experiments. The polymer-based fabrication strategy will enable fast, low-cost device fabrication, as well as allowing for direct imaging of samples, in situ spectroscopy, and laser-based triggering of reactions for time-resolved X-ray crystallography studies. Aim 3: Develop fixed-target platforms for chemical triggering of reactions for time-resolved serial crystallography analysis. We will develop strategies to enable the controlled addition of a chemical species (i.e., substrate, pH change) to enable the chemical triggering of reactions in crystals. Aim 4: Utilize graphene-based devices to study structural dynamics based on high voltage triggering. We will integrate graphene-based circuitry into our microfluidic devices to enable the use of a voltage-jump as both a general strategy for triggering protein motions and to control electron transfer reactions. The team is well qualified, merging expertise in microfluidics, X-ray science, and structural biology, with an established history of developing new technology to address challenges within the structural biology community. The impact will be improvements in the ability to perform time-resolved s...