Microscale vapour diffusion for protein crystallization

Abstract

The development of new crystallization platforms via the application of high-throughput technologies has delivered a plethora of crystallization plates suitable for robot-driven and manual setups. However, practically all these plates (except for microfluidic channel chips) are based on a very similar design and well (precipitant):drop (protein) volume ratios. A new type of crystallization plate (microplate) has therefore been developed and tested that still employs the classical vapour-diffusion technique but minimizes the precipitant well volume to 1.2 microl for a 150 nl protein drop setup. This enables a very significant saving on the total bulk of the crystallization screen, hence allowing the application of new, rare and expensive solutions in automated crystallization-screening procedures. Additionally, owing to the very low drop:well volume ratio, the new microplate can significantly accelerate the equilibrium time necessary for crystal nucleation and growth, in many cases shortening the high-throughput crystallization screening process to a few hours.

An Overview of Biological Macromolecule Crystallization

“…This technique utilizes evaporation and diffusion of water (and other volatile species) between a small droplet (0.5–10 μL), containing protein, buffer and precipitant, and a reservoir (well), containing a solution with similar buffer and precipitant, but at higher concentrations with respect to the droplet [76–78]. The wells are sealed by creating an interface of vacuum grease between the rim of each well and the cover slip, or by using, in specific cases, a sealing tape.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…In particular, COC has seen widespread adoption as the polymer film of choice for X-ray compatible devices, 46 including simple channel structures for counterdiffusion, 56,58,60,61,126 droplet-based devices, 108,109,118 and larger-scale X-ray compatible wellplates. [127][128][129][130][131][132][133][134][135][136][137][138][139][140] However, further decreasing the device thickness to achieve the signal-to-noise levels required for microcrystallography is a significant materials' challenge. Typical reports of X-ray compatible microfluidics describe results where the path length of the device materials is nearly twice that of the crystal of interest.…”

Higher-throughput approaches to crystallization and crystal structure determination

“…In a recent development, a new microplate has been designed and tested in which the size of the reservoir solution is reduced to 1.5 μl. This is especially desirable where the precipitant solutions are precious [10], for example if they contain expensive or scarce polymers, such as those being developed for the crystallization of membrane proteins. Furthermore, because of the smaller volumes, the new plates reduce the time required for crystal nucleation and growth, in some cases shortening the high-throughput crystallization screening process to a few hours.…”