Abstract
Serial synchrotron crystallography (SSX) is enabling the efficient use of small crystals for structure-function studies of biomolecules and for drug discovery. An integrated SSX system has been developed comprising ultralow background-scatter sample holders suitable for room and cryogenic temperature crystallographic data collection, a sample-loading station and a humid `gloveless' glovebox. The sample holders incorporate thin-film supports with a variety of designs optimized for different crystal-loading challenges. These holders facilitate the dispersion of crystals and the removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or the risk of generating saturating Bragg peaks, are compatible with existing infrastructure for high-throughput cryocrystallography and are reusable. The sample-loading station allows sample preparation and loading onto the support film, the application of time-varying suction for optimal removal of excess liquid, crystal repositioning and cryoprotection, and the application of sealing films for room-temperature data collection, all in a controlled-humidity environment. The humid glovebox allows microscope observation of the sample-loading station and crystallization trays while maintaining near-saturating humidities that further minimize the risks of sample dehydration and damage, and maximize working times. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed-orientation and oscillation data collection. Its ease of use, flexibility and optimized performance make it attractive not just for SSX but also for single-crystal and few-crystal data collection. Fundamental concepts that are important in achieving desired crystal distributions on a sample holder via time-varying suction-induced liquid flows are also discussed.
Highlights
As X-ray crystallography has passed its centenary (Bragg, 1912) and >133 000 macromolecular crystallographic structures have been deposited in the Protein Data Bank, the field of macromolecular crystallography (Rupp, 2009) continues to evolve
In experiments on FMX using fluoroacetate dehalogenase (FAcD) samples cooled to 100 K, the sample support was rastered relative to the beam in steps of 20 mm, and 5 of oscillation data were collected at each position
The modular sample-support configuration and an efficient pipeline for the microfabrication of sample-support films facilitated cycles of design, testing and optimization, and yielded insights into how sample supports can best be used with diverse crystals
Summary
As X-ray crystallography has passed its centenary (Bragg, 1912) and >133 000 macromolecular crystallographic structures have been deposited in the Protein Data Bank (https:// www.rcsb.org/), the field of macromolecular crystallography (Rupp, 2009) continues to evolve. Key aspects of current fixed-target serial microcrystallography, including the use of ultrathin substrates with arrays of holes and liquid removal by back-side blotting to minimize background scatter, were demonstrated 14 years ago using MiTeGen MicroMeshes, which were initially developed to enable data collection from hundreds of 5–12 mm cypovirus polyhedra crystals (Coulibaly et al, 2007). Compared with moving-target approaches, fixed-target approaches typically allow data collection from a larger fraction of the available crystals, with some methods approaching 100% hit rates (Oghbaey et al, 2016), and at SR sources crystal oscillation during exposure further reduces the number required for a complete data set. We have developed an integrated fixed-target SSX samplepreparation and handling system, based in part on previously demonstrated concepts, that is and inexpensively manufactured, is highly customizable for different crystal morphologies and crystal-handling challenges, has excellent X-ray and optical performance, is suitable for both RT and cryo-SSX, and is compatible with the existing infrastructure for high-throughput mail-in cryocrystallography
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