Homogeneous batch micro-crystallization of proteins from ammonium sulfate

Abstract

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 α-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.

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

“…SSX experiments benefit from homogenous micro-crystals. Strategies have been proposed to reproducibly achieve this by modifying the conditions used to produce single well ordered crystals that have been traditionally the focus of many crystallographic laboratories (Tenboer et al, 2014;Beale et al, 2019;Stohrer et al, 2021). Although the correct understanding of the crystallization phase diagram is undoubtedly beneficial to facilitate the macrocrystal-to-microcrystal transition, the complex nature of biological crystal formation suggests that this can be a time-consuming process.…”

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

“…Once successful crystallization conditions are determined, optimization of the latter according to the general phase behaviour of proteins could lead to faster crystallization rates suitable for this approach. The focus of protein crystallization research has been, obviously, on the reliable production of large, well diffracting crystals, with few examples focussing on the directed generation of small crystals for solid state NMR (Martin and Zilm, 2003), SFX and SSX (Beale et al, 2019; Stohrer et al, 2021; Tenboer et al, 2014). The JINXED method could even be used to screen for crystallization conditions and crystallization times without the need for visual assessment and direct proof of crystalline properties.…”

“…The screening for suitable crystallization conditions may be time-consuming and extensive, but this challenge is equally faced in protein crystallography in general (Chayen, 2004;Chayen and Saridakis, 2008).Once successful crystallization conditions are determined, optimization of the latter according to the general phase behaviour of proteins could lead to faster crystallization rates suitable for this approach. The focus of protein crystallization research has been, obviously, on the reliable production of large, well diffracting crystals, with few examples focussing on the directed generation of small crystals for solid state NMR (Martin and Zilm, 2003) , SFX and SSX (Beale et al, 2019;Stohrer et al, 2021;Tenboer et al, 2014).…”

Best practices for time-resolved serial synchrotron crystallography

“…Batch methods, in which large volumes (usually >50 ml) of both mother liquor and protein solution are mixed in a vial, are ideal for obtaining large numbers of homogenously sized microcrystals. Recently, a number of systematic approaches for obtaining microcrystals via batch-crystallization methods using different precipitation agents have been described (Beale et al, 2019;Stohrer et al, 2021). An extension to batch crystallization takes advantage of rapid evaporation from a vacuum environment (Martin & Zilm, 2003).…”