Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
Serial femtosecond
crystallography (SFX) is a powerful technique
that uses X-ray free-electron lasers (XFEL) to determine structures
of biomolecular complexes. Specifically, it benefits the study of
atomic resolution structures of large membrane protein complexes and
time-resolved reactions with crystallography. One major drawback of
SFX studies with XFELs is the consumption of large amounts of a protein
crystal sample to collect a complete X-ray diffraction data set for
high-resolution crystal structures. This increases the time and resources
required for sample preparation and experimentation. The intrinsic
pulsed nature of all current X-ray sources is a major reason why such
large amounts of sample are required. Any crystal sample that is delivered
in the path of the X-ray beam during its “off-time”
is wasted. To address this large sample consumption issue, we developed
a 3D printed microfluidic system with integrated metal electrodes
for water-in-oil droplet generation to dynamically create and manipulate
aqueous droplets. We demonstrate on-demand droplet generation using
DC potentials and the ability to tune the frequency of droplet generation
through the application of AC potentials. More importantly, to assist
with the synchronization of droplets and XFEL pulses, we show that
the device can induce a phase shift in the base droplet generation
frequency. This novel approach to droplet generation has the potential
to reduce sample waste by more than 95% for SFX experiments with XFELs
performed with liquid jets and can operate under low- and high-pressure
liquid injection systems.
Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.
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