Microbatch Mixing: “Shaken not Stirred”, a Method for Macromolecular Microcrystal Production for Serial Crystallography

Abstract

The advances of serial crystallography techniques at synchrotron and X-ray free electron laser facilities have made possible the acquisition of useable data sets to determine 3-dimensional structures of macromolecules from micro- to nanosized crystals. In addition, the same technological hallmarks have contributed significantly to the field of time-resolved crystallography. However, the production of usable crystalline slurries for serial crystallographic experiments has been one of the limiting factors and contributes to an alternative sample “bottleneck” in crystal growth. In this study, we propose a method: labeled microbatch mixing (MBM), which has the capability to produce large quantities of microcrystals of macromolecules suitable for serial crystallographic experiments. This is shown to be successful for producing lysozyme, carbonic anhydrase, and adeno-associated virus crystals. MBM takes advantage of secondary nucleation induced by mixing via the application of steady agitation during the crystallization process. This leads to excessive nucleation, resulting in large quantities of well-diffracting microcrystals. MBM therefore presents a method that can potentially be applied to a range of macromolecules and a possible simple protocol to produce microcrystals for serial crystallographic experiments.

Carbonic anhydrase II microcrystals suitable for XFEL studies

“…precipitant solution. In a 24-well culture plate, 5 ml seed stock, 300 ml CA II (30 mg ml À1 ) and 1200 ml precipitant solution were added to each well as described previously (Mahon et al, 2016). Microcrystal growth was observed after 12 h. The microcrystal suspension was diluted in precipitant solution (1:4 ratio) and syringe-filtered through a metal filter, removing crystals of greater than 100 mm in length.…”

A Bright Future for Serial Femtosecond Crystallography with XFELs

“…Crystals grown in solution are most often initially obtained by high-throughput screening in plates and then optimized for batch setups (either in plates or in vials) which are then injected into the XFEL beam in their native mother liquor [70–72]. Crystals grown in solution can easily be concentrated by centrifugation and then filtered prior to injection thereby removing large crystals and impurities to avoid clogging the injector.…”

Protein crystallization: Eluding the bottleneck of X-ray crystallography

“…Airborne contamination and other variables are blocked from contacting with the sample reducing interference in protein crystallization [ 2 , 14 ]. Batch crystallization is also useful for producing large quantities of microcrystals suitable for serial crystallographic experiments [ 17 ].…”