See how MRC scientists can quickly and directly optimize macromolecular crystallization conditions

Introduction

The X-ray crystallography method is currently the most effective means of obtaining biomacromolecular structures. But getting crystals from purified proteins, nucleic acids, or macromolecular complexes is a huge challenge. Along with the lengthy experimental setup and process, often a very low experimental success rate. First, the investigator will use a commercially available kit to perform a preliminary screening of the crystallization conditions for a purified sample over a wide range. Then, a round of optimization screening is performed according to the specific experimental conditions to repeat and improve the results obtained in the preliminary screening. By optimizing the conditions and obtaining high quality crystals, reliable X-ray diffraction data can be obtained, from which the high resolution structure can be resolved.

Typically, optimized screening is done by hand using a conventional liquid applicator. The whole process can be very time consuming. In addition, commonly used stock solutions contain low surface tension or high viscosity components such as PEGs. These factors often lead to inaccurate loading and cross-contamination.

Scientists at the MRC Molecular Biology Laboratory used the dragonfly, a fully automated liquid handling workstation developed by TTP Labtech in the UK, to easily overcome the above obstacles. The fully automated optimization resulted in the crystallization of three different protein samples. This innovative instrument uses solid-phase displacement technology and non-contact loading principle to eliminate cross-contamination and to achieve a 1 μL loading accuracy for all types of liquids, including extremely viscous glycerol. CV values ​​are controlled within 5% to ensure accurate loading. Researchers can quickly and easily prepare the screening conditions they need in any SBS template. A sample plate containing 96 screening conditions can be prepared in 3-6 minutes.

Experimental materials and methods

Protein samples of Lysosyme, Concanavalin A, and Catalase were prepared as reported and a preliminary screen was completed.

The concentration of the precipitant (PEG) and the additive (eg, propanediol) is increased or decreased in a diagonal gradient according to the preliminary screening conditions obtained for the different proteins, thereby further optimizing the screening conditions. Such a sample loading method can produce a concentration gradient of two different reagents in the crystallizing plate, and different liquid storage amounts to be added in each well are automatically and sequentially distributed. Each protein will try three different pH conditions, and nine samples containing different screening conditions will be prepared with dragonfly.

The 9 screening plates were placed on a horizontal shaker and mixed, and the mixture of the protein solution and the screening solution was directly applied (100 nL + 100 nL). The culture was observed daily, and a photograph of the grown crystal was obtained three weeks later.

result

The preparation time for each optimized screening plate is no more than 4.5 minutes. After 7 days of culture, crystal growth was observed in 3 to 15 wells per plate.

The conditions for crystal nucleation and growth are optimized by adjusting the concentration and pH of the solution. In this way, a large number of single large volume crystals are obtained. In the course of structural analysis and ligand binding, severe crystal loss often occurs. The acquisition of a large number of crystals can overcome the bottleneck of this research.

summary

Dragonfly has been shown to perform crystallization conditions in 96 wells quickly and efficiently. Compared to traditional manual loading, the speed of the dragonfly preparation to optimize the screening conditions is increased by at least 3 times.

The solid phase displacement and non-contact sample loading technology used by the dragonfly ensures the accuracy of the loading of any viscosity liquid, and also eliminates the occurrence of cross-contamination. The setup and operation of the instrument is very simple and convenient, and different users can perform independent experimental operations without complicated training. With the success of the experiment, the use of dragonfly to optimize the screening conditions has become a daily experimental tool for screening in the MRC Molecular Biology Laboratory. Enables MRC scientists to quickly and directly optimize macromolecular crystallization conditions.

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