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Acta crystallographica / Section D 77(2), 194-204 (2021). doi:10.1107/S2059798320015454, The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate $��$-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments., Published by Wiley, Bognor Regis

A 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) has been developed and used to rapidly collect highly redundant diffraction data sets from protein microcrystals in flow using a monochromatic serial synchrotron crystallography approach., Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.