Hanging drop, crystallization method

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Figure 3 Protein crystallization diagrammatic representation of the hanging-drop method of vapor diffusion.

As described earlier, there are a number of different ways of crystallizing proteins. By far, the most common approach is the vapor-diffusion method, as mentioned earlier. Approximately, 70% of the crystal structures reported have been crystallized through variations of the vapor-diffusion method. The technique can be carried out in a number of ways the simplest two being the hanging drop method, and the sitting drop ... 

Although microbatch is the simplest method of crystallization, it is a relatively new technique and many experimenters still prefer to use vapour diffusion which has been around and has worked well for over 40 years. Hence, there has also been major development in automating and scaling down the quantities of sample using the popular vapour diffusion method (both sitting and hanging drops). An increasing variety of robots are available commercially. 

The main technique employed to set up crystal screens is the vapour diffusion method, either in the hanging drop or sitting drop set up. This method is based on slowly concentrating the droplet solution against a reservoir solution of infinite volmne (ml scale) compared to the volume of the droplet ( xl scale, see Fig. 14.2). Other techniques based on diffusion or counter-diffusion in agarose gels (Biertmnpfel et al., 2002) can also be useful. The... 

In the example of the aminoglycoside/ A site complexes, different crystallization solutions were prepared to test various glycerol/MPD ratios 5, 2, 1, 0.75, 0.67, and 0.5 (Table 14.2). All trials are performed at the optimal temperature of 37°C using the vapour diffusion method in the hanging drop set-up 1 xl RNA-antibiotic complex solution was added to 1 xl crystallization solution and equilibrated over a 40% MPD reservoir. 

Mikol, V, Rodeau, J.-L. and Giege, R. (1990). Experimental determination of water equilibration rates in the hanging drop method of protein crystallization. Anal. Biochem. 186, 332-339. 

The technique of choice for screening crystallization conditions is the vapour diffusion hanging drop described in Chapter 3. in this method drops containing different concentrations of protein-DNA mixtures, buffer, additive, and precipitant are suspended from a sihconized coverslips placed over sealed reservoirs containing different precipitant... 

Figure 3.2 Growing crystals by the hanging-drop method. The droplet hanging under the cover slip contains buffer, precipitant, protein, and, if all goes well, growing protein crystals.

One widely used technique is vapor diffusion, in which the proteinlprecipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. An example of this technique is the hanging-drop method (Fig. 3.2). 

Crystallization—Small crystals (0.05 X 0.1 X 0.1 mm) were obtained using the hanging drop/vapor equilibrium method (18). 10-pl drops of 2.5 mg/ml ALBP in 0.05 M Tris, 60% ammonium sulfate, 1 mM EDTA, 1 mM dithiothreitol, 0.05% sodium azide buffer with a pH of 7.0 (crystallization buffer) were suspended over wells containing the same buffer with varying concentrations of ammonium sulfate, from 75 to 85% saturation. Small, well shaped crystals were formed within a month at an 80% saturation and 19 crystallization buffer over a well containing the same buffer. Large crystals,... 

Before crystallization trials, the protein was subjected to gel filtration on Superdex-75 (Pharmacia) in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 50 mM 2-mercaptoethanol, 150 mM sodium chloride, 5% glycerol and 5% 2-propanol, as described previously (12). The statine-based inhibitor, LP-149 (Ac-Nal-Val-Sta-Glu-Nal-NH2 e Nal is naphtylalanine and Sta is statine) (Fig. 1), was prepared at Lilly Research Laboratories (K. Hui, unpublished results). Crystallization was carried out at 4 °C using the hanging-drop vapor diffusion method as follows 2.5 //I of the FIV PR(D30N) at 7 mg/ml complexed with LP-149 (1 4 molar ratio) in 50 mM imidazole-HCl pH 7.0 containing ImM EDTA and 1 mM dithiothreitol were mixed with an equal volume of 2 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 (Hampton Crystal Screen, solution 47). Crystals appeared within a few days and reached the size of 0.2 x 0.2 X 0.4 mm in one week. 

FIGURE 2.8. Some methods for growing crystals from solution, (a) Slow solvent evaporation, primarily used for small molecules, (b) Vapor diffusion, (c) The hanging drop method, primarily used for macromolecules, (d) A crystallization plate used for hanging drops there are slightly different crystallization conditions in each well, varied systematically with respect to, for example, pH and ionic strength. 

Crystals of PEPC were grown by the hanging drop vapor diffusion method as... 

We therefore describe the basis of macromolecular crystallography and provide a summary of how to understand the results of a crystallographic experiment. We start with a mathematical description of what a crystal means in terms of symmetry this applies to all crystals, whether macromolecular or not. Later, we describe how protein crystals grow by using the hanging drop and sitting drop vapor diffusion methods this explains why protein crystals are so fragile and scatter X-rays very weakly. 

FIGURE 30.8 Principle of the method of vapor diffusion in hanging drops. A droplet (0.1-1 pU) of a solution of protein or protein complex is mixed with crystallization buffer (typically in a 1 1 ratio), suspended above a reservoir containing the crystallization buffer ( 0.1-0.5mL) and allowed to equilibrate. Because of the concentration gradient, water is transferred from the drop to the reservoir via the vapor phase. As a result, the concentrations of protein and precipitant in the drop increase. If a supersaturated state is reached, the protein will then precipitate. The precipitate is usually amorphous, but crystals will form in successful experiments. 

Fig. 1. The vapor diffusion method for growing protein crystals. Water equilibrates from the more dilute protein solution in the drop into the more concentrated solution at the bottom of the beaker. This concentrates the solution slowly in the hanging drop.

Figure 2.7 The seeding method A small crystal is washed in a series of solutions in which it slowly dissolves, e.g. solutions with a decreasing precipitant concentration. In this way the surface of the crystal is etched and cleaned. It is then transferred to a fresh drop of protein solution with so high a concentration of precipitating agent that the crystal does not dissolve. The drop is then equilibrated with a more concentrated precipitating solution in either the hanging drop or sitting drop mode. Figure kindly supplied by J. Drenth, University of Groningen and reproduced with permission.

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