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High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.