Abstract
Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.
Highlights
Proteins are central to all aspects of cellular life, from metabolism to facilitated diffusion across membranes, to defense against foreign particles
Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states
The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals
Summary
Proteins are central to all aspects of cellular life, from metabolism to facilitated diffusion across membranes, to defense against foreign particles. A proof-of-principle mixing injector, developed by Wang et al, was a variation of the GDVN with the central sample line replaced by coaxial supply lines for crystal solution and reactant solution.21 Within this device, the two solutions mixed by diffusion prior to focusing into a free liquid jet. The low hit rate may prevent the collection of sufficient diffraction patterns to solve a structure over the course of a normal XFEL experiment This device has a nominal submillisecond mixing time for some reactants; this calculation only considers the diffusion time after hydrodynamic focusing and neglects time the protein is in contact with the substrate before full focusing occurs, which could be much longer than the mixing time due to low sample velocity and slow focusing.
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