Project Summary/Abstract One of the new frontiers in structural enzymology is the expansion from a three-dimensional to a truly four- dimensional approach by adding the time dimension to structural studies. While Synchrotron Radiation (SR) crystallography and cryo Electron Microscopy allow the determination of structures in minute detail they are in most cases performed on frozen static samples. With the advent of X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at Stanford, and the development of the “probe before destroy” concept it now is possible to follow structural changes in enzymes in real time and under close to physiological conditions at room temperature (RT). Driven by the success of XFELs and recent advances in detector technology and storage ring and beam line design, several SR sources are also starting to offer time resolved crystallography at RT. These unprecedented capabilities will open new fields of research, not only in biomedical sciences but also in many other areas. Due to the “probe before destroy approach” utilized here, the samples generally need to be replaced after a single X-ray exposure. As biological samples of interest are often only available in scarce amounts, it is mandatory to develop a robust method to introduce the sample into the X-ray interaction region in a continuous manner that minimizes the required sample amount. In order to obtain true “molecular movies” of enzymes of biomedical importance in action, which will contribute to a deeper mechanistic understanding of these molecular machines, it is essential to synchronize the enzyme in the probed sample volume and initiate the reaction of interest in a temporally well-defined manner. Methods for reaction initiation can include mixing with a substrate/chemical compound, or utilize other stimuli such as light, temperature jump, or change in pH or electrical potential. In the frame of this proposal, we will continue the development of robust and versatile sample delivery and reaction triggering methods. We will also integrate multi-modal detection methods, combining X-ray diffraction with complementary in situ spectroscopic techniques to probe both global structures and chemical properties of enzymes concurrently. We will focus on the development of drop-on-demand methods based on acoustic transducer technology, but also explore other droplet dispensing technologies and microfluidics to substantially diminish/eliminate any sample wastage. We will improve the previously developed prototypes for depositing the drops on a moving support, such as a tape or wheel, that can circulate and is self-cleaning, for non-stop continuous operation at the XFEL or SR facility. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-gas and liquid-liquid mixing, including with micron size droplet collision methods to achieve faster time resolution. Experiments on well-defined enzyme model systems will be accommodated...