Administrative Supplement for Structural Dynamics in Biology Resource Year 2

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Project Summary – Administrative Supplement Request – 1P41GM139687-02 – Sebastien Boutet (PI). This administrative supplement request is driven by overall goals and aims of the Structural Dynamics in Biology BTRR at SLAC National Accelerator Laboratory. The BTRR is aimed at enhancing and developing the unique capabilities of the SLAC Linac Coherent Light Source (LCLS) for biomedical applications. The requested supplement will support a broad user base and further the goals of the DBPs and the TR&Ds. By overcoming the limitations of radiation damage at the synchrotron, LCLS has been particularly impactful in the study of large macromolecular machines that form small crystals with high solvent content, making them delicate, difficult to cryo-preserve and extremely radiation sensitive. For example, a major breakthrough was the application of SFX to examine crystals of intrinsic membrane proteins, such as GPCRs, grown in LCP. GPCRs are the largest group of targets for drug development, used to treat a wide variety of illnesses (e.g. cancer, cardiovascular disease, and mental illness). LCLS is also impactful in the study of metalloenzymes, which are critical to nearly all biological processes and as such represent a rich target space for therapeutics development. High-resolution structural studies of metalloproteins are particularly challenging at the synchrotron because the metal centers, especially those that are redox active, are very susceptible to x-ray induced photoreduction. Further, the ultrafast (~40 fs) x-ray pulses produced by the LCLS open new possibilities in directly observing dynamic processes involved in macromolecular function. Moreover, many of the P41-derived developments that enable the rapid collection of data using multiple small crystals, are applicable both at LCLS and at the synchrotron to mitigate radiation damage. By expanding the capabilities at LCLS and at the SSRL synchrotron, the BTRR opens more macromolecular machines to structural characterization, including time- resolved studies over a wide range of biomedically relevant time scales. Integrating with, and enhancing the existing programs at SSRL and LCLS, the BTRR will provide support, expertise and training to the broad biomedical community. This administrative supplement will enhance and expand the BTRR capabilities that are provided to general users and to the P41 driving biomedical projects. The acquisition of a high speed x-ray chopper will fill a critical need of the BTRR for efficient use of SSRL BL12-1 with small crystals delivered by liquid/crystal injectors and for time-resolved measurements. In addition to achieving microsecond time resolution, by breaking up the continuous x-ray beam into short pulses, the chopper ensures crystals delivered by injectors are not destroyed by x-ray damage before they are fully translated into the x-ray beam position, enabling exposure to unattenuated monochromatic or pink-beam at BL12-1. In addition, this supplement requests a biocompatible resin and ceramic 3D printer with 2-micron resolution that will allow for rapid prototyping and optimization of sample injectors that will provide reliability and ease that will broaden the user community. Finally, the proposal also requests a system to measure the conductivity of samples in an automated manner which will allow to further the goals of the BTRR in characterizing sample conditions and mapping those onto the ideal sample delivery method to be used for a given sample, providing users with structured decision-making on how to best perform their experiment. This will complement ongoing efforts in physicochemical characterization by adding conductivity, an important parameter for electrokinetic sample delivery, to lookup tables to be published.