RESUMO
The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around "workhorse" for serial crystallography measurements. This Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system.
RESUMO
Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70â Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.