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-
- Award ID(s):
- 1943448
- NSF-PAR ID:
- 10316210
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Date Published:
- Journal Name:
- Journal of Applied Crystallography
- Volume:
- 55
- Issue:
- 1
- ISSN:
- 1600-5767
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract manno -octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported. -
High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1107/S1600576721011079
