Crystallization hanging drop

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.)...

It proved possible to crystallize the Fepr protein over a wide range of pH, 5.9-8.0, and PEG 8000 concentrations, using both sitting and hanging-drop techniques. The crystals used for the X-ray analysis were produced using the following procedure (29) ... 

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. 

Dilution experiments involve revisiting the crystallization drops which may cause disruption to the trial. An alternative way of backing off without touching the trial drop can be achieved in hanging drops in the following way the coverslips holding the drops are incubated for some time over reservoir solutions that normally give many small crystals (Protocol 3.6). 

Figure 3.6 Application of an oil barrier to improve crystals in hanging drops, (a) A standard hanging drop trial (b) a trial containing an oil barrier. Modified from Chayen (1997b). J. Appl. Cryst. 30, 198-202, with permission from the lUCr.

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... 

Schwartz, A. M., and Berglund, K. 2000. In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop. J. Cryst. Growth 219 753-60. 

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,... 

Schwartz et al. evaluated the use of fibre-optic probe dispersive Raman and PLS calibration to study crystallisation in a hanging drop experiment. They could show that the lysosome concentration could be monitored during the experiment through the concentration phase, nucleation and crystal growth [27], In later experiments, they were able to utilise in-line Raman data to control the concentration of lysozyme to affect the crystallisation path and thereby obtaining the desired large single crystals [28], The same setup was used for crystallisation of apoprotein. Also in this case the Raman data were used to control the evaporation and thus the supersaturation for optimised crystal size [29]. 

Crystals were grown by vapor diffusion in hanging drops from solutions containing methylpentanediol (MPD), polyethylene glycol) (PEG), ammonium sulfate (AS), or low-molecular-weight alcohols (A). 

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. 

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