Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers

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.

Watching proteins function with time-resolved x-ray crystallography

“…Typically, this number is small, on the order of 5 to 10%. In favorable cases, however, the extent of reaction initiation can be as high as 40 to 50%, even 100% in favorable cases (Šrajer et al ., 2001; Knapp et al ., 2009; Tenboer et al ., 2014; Barends et al, 2016). …”

“…With these tiny crystals there are two challenges, (i) the mixing times might become longer than the diffusion times, and (ii) the crystals are so small that with conventional methods diffraction pattern cannot be collected due to radiation damage. The first challenge can be overcome with specially designed liquid-liquid mixing injectors (Wang et al ., 2014; Calvey et al ., 2016) reminiscent of GDVNs, where a stream of microcrystals is rapidly mixed with a concentric flow of substrate.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…29,30,35,220 By including an additional capillary into the GDVN architecture, diffusive mixing between the crystal slurry and an outer sheath of triggering solution can be achieved. The time for mixing can then be altered by adjusting the location of the mixing point within the larger GDVN structure.…”

“…[19][20][21][22][23][24][25][26][27][28][29][30] However, these large-scale serial methods suffer from the need to manipulate crystals and/ or from inefficient sample utilization. 3,[31][32][33][34][35][36] The material-intensive nature of these experiments extends the long-standing challenge of growing a well-diffracting crystal to reproducibly generating a large number of high quality, isomorphous crystals. These issues are then further compounded by the need to deliver those samples efficiently to the X-ray beam.…”

Fluid sample injectors for x-ray free electron laser at SACLA

“…A mix-and-jet injector enables rapid mixing of a reactant solution and a crystal suspension in a timescale of millisecond for capturing protein motion initiated by chemicals [33,34] .…”