Protein crystallization for genomics: towards high-throughput optimization techniques

Abstract

Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X‐ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X‐ray analysis and bioinformatics stages once diffraction‐quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large‐scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high‐throughput techniques.

The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

“…Studies on biocatalysis showed that some imidazolium-based ILs could enhance protein activity and make it more stable [21]. In 2002, scientists found gel media, in some way mimicking a microgravity environment, could reduce density driven convection and sedimentation, which resulted in a significant improvement of crystal order [22,23]. Chayen [24,25] reported that paraffin and silicone oils could prevent the evaporation of small-volume microbatch trials.…”

Succeeding with seeding: some practical advice

“…Although efforts to automate crystal optimization are now underway [1], a follow-up optimization experiment is often still designed and implemented manually. Microseeding, and especially streak seeding, are easy to perform which makes them attractive as optimization methods.…”

“…However, screening in itself, no matter how extensive, is still no guarantee that crystals will be found or that they will provide diffraction-quality data. At least half of the crystals obtained in an initial screen cannot be used without further optimization [1]. Therefore, optimization methods are often crucial for the success of a crystallization project.…”

Investigation of molecular condensation on air-liquid interface for protein crystallization

“…Crystallization using micro-bubble Generally, one of the conventional protein crystallization methods is hanging drop method and sitting drop method which use supersaturation of the protein solution and classified as vapor diffusion [6][7] In case of the method, a drop composed of protein and regent is placed in the arrayed well chamber with reservoir solution. Typically, the drop contains a lower reagent concentration than that of the reservoir solution.…”