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1. Segmented flow generator for serial crystallography at the European X-ray free electron laser

   https://doi.org/10.1038/s41467-020-18156-7  

   Echelmeier, Austin ; Cruz Villarreal, Jorvani ; Messerschmidt, Marc ; Kim, Daihyun ; Coe, Jesse D. ; Thifault, Darren ; Botha, Sabine ; Egatz-Gomez, Ana ; Gandhi, Sahir ; Brehm, Gerrit ; et al ( September 2020 , Nature Communications)

   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 immisciblemore »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.

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2. High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

   https://doi.org/10.1107/S2052252519011655  

   Moreno-Chicano, Tadeo ; Ebrahim, Ali ; Axford, Danny ; Appleby, Martin V. ; Beale, John H. ; Chaplin, Amanda K. ; Duyvesteyn, Helen M. ; Ghiladi, Reza A. ; Owada, Shigeki ; Sherrell, Darren A. ; et al ( November 2019 , IUCrJ)

   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 differencemore »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.« less
3. Lifetimes and spatio-temporal response of protein crystals in intense X-ray microbeams

   https://doi.org/10.1107/S2052252517013495  

   Warkentin, Matthew A. ; Atakisi, Hakan ; Hopkins, Jesse B. ; Walko, Donald ; Thorne, Robert E. ( November 2017 , IUCrJ)

   Serial synchrotron-based crystallography using intense microfocused X-ray beams, fast-framing detectors and protein microcrystals held at 300 K promises to expand the range of accessible structural targets and to increase overall structure-pipeline throughputs. To explore the nature and consequences of X-ray radiation damage under microbeam illumination, the time-, dose- and temperature-dependent evolution of crystal diffraction have been measured with maximum dose rates of 50 MGy s −1 . At all temperatures and dose rates, the integrated diffraction intensity for a fixed crystal orientation shows non-exponential decays with dose. Non-exponential decays are a consequence of non-uniform illumination and the resulting spatial evolution of diffracted intensitymore »within the illuminated crystal volume. To quantify radiation-damage lifetimes and the damage state of diffracting crystal regions, a revised diffraction-weighted dose (DWD) is defined and it is shown that for Gaussian beams the DWD becomes nearly independent of actual dose at large doses. An apparent delayed onset of radiation damage seen in some intensity–dose curves is in fact a consequence of damage. Intensity fluctuations at high dose rates may arise from the impulsive release of gaseous damage products. Accounting for these effects, data collection at the highest dose rates increases crystal radiation lifetimes near 300 K (but not at 100 K) by a factor of ∼1.5–2 compared with those observed at conventional dose rates. Improved quantification and modeling of the complex spatio-temporal evolution of protein microcrystal diffraction in intense microbeams will enable more efficient data collection, and will be essential in improving the accuracy of structure factors and structural models.« less
4. Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures

   https://doi.org/10.1107/S1600576720013503  

   Doukov, Tzanko ; Herschlag, Daniel ; Yabukarski, Filip ( December 2020 , Journal of Applied Crystallography)

   Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure–function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described providesmore »complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09–1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.« less
5. Co-flow injection for serial crystallography at X-ray free-electron lasers

   https://doi.org/10.1107/S1600576721011079  

   Doppler, Diandra ; Rabbani, Mohammad T. ; Letrun, Romain ; Cruz Villarreal, Jorvani ; Kim, Dai Hyun ; Gandhi, Sahir ; Egatz-Gomez, Ana ; Sonker, Mukul ; Chen, Joe ; Koua, Faisal H. ; et al ( February 2022 , Journal of Applied Crystallography)

   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 andmore »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.« less