Project Summary/Abstract The advent of the X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at Stanford has the potential to revolutionize the field of structural studies of biological systems. It is now possible using the XFELs to determine the structures of enzymes and follow their reactions in real time, at room temperature. These unprecedented capabilities will open new fields of research, not only in biological sciences but also in other areas. One bottle-neck in the use of the XFELs has been the lack of a robust method to introduce the sample into the X-ray interaction region in a continuous manner as the samples are destroyed after exposure to just one pulse of X-rays, and at the same time minimize the amount of biological sample used, which are often only available in small quantities. Moreover, it is imperative to be able to trigger the reactions, by some method, such as using a substrate/chemical compound, or some other stimulus such as light, or by a temperature jump, or electrical potential so that we can follow the reaction as it happens in real time, in order to be able to understand how the enzyme functions. This proposal deals with exploring and constructing robust and versatile sample delivery and reaction triggering methods. Also, when combined with complementary techniques like spectroscopy and other methods in situ, both global structures and chemical properties of enzymes can be obtained concurrently, providing insights into the interplay between the protein structure/dynamics and chemistry at an active site. We will focus on the development of drop-on-demand methods, mostly based on an acoustic transducer, that will substantially diminish or eliminate any sample wastage which is a problem with the more commonly used capillary based sample delivery methods that also have other problems such as clogging that are eliminated with an acoustic droplet ejector. We propose to develop methods 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. There we can use the X-ray pulses to study the intermediate states in enzymes that will be generated by substrate (liquid/gas) activation, which covers most of the enzymatic reactions. Other triggering methods such as light, or temperature jump, or an electric potential that will be used to study redox active enzyme systems, will be built-in into our sample delivery system. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-liquid mixing with micron size droplet collision methods to achieve faster time resolution that can be followed by subsequent time- evolution before the X-ray probe.