Sitting drops

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

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 ... 

Harvesting crystals from microbatch is slightly more difficult than harvesting from coverslips or from standard sitting drops (Protocol 3.3). However, after... 

Soriano, T. M. and Eontedlla-Camps, J. C. (1993). Astec -an automated-system for sitting-drop protein crystallization. J. Appl. Cryst. 26,558-562. 

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... 

Harlos, K. (1992). Micro-bridges for sitting drop crystallisations. J. Appl. Cryst. 25,536-538. 

The first crystal structure of CKX, namely ZmCKXl, was published in 2004 [147], Fig. (4A). So far it is the only protein from the number of different enzymes and receptors acting specifically on cytokinins, where the structure has been solved. The crystals of ZmCKXl, expressed in the yeast Pichia pastoris, were crystallized by a sitting drop vapor diffusion method and grown in 100 mM sodium acetate (pH 4.6), 200 mM ammonium sulfate, and 12% (w/v) PEG 5.000 monomethylether. Crystals belonged to the P492i2 space group. 

Since polyethylene glycol) solutions are not volatile, this precipitant must be used like salt and equilibrated with the protein by slow mixing or vapor equilibration. This latter approach, utilizing either hanging drops over 0.5 ml reservoirs, or sitting drops in plastic plates, has proved the most popular. When the reservoir concentration is in the range 5% to 20%, the protein solution to be equilibrated should be at an initial concentration of about half of that, which is conveniently obtained by adding an equal volume of the reservoir to that of the protein solution. 

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 5 Setups for vapor diffusion, (a) Hanging drop vapor diffusion, (b) Sitting drop vapor diffusion in a modern 96-well plate. In both cases, a greased coverslip seals the well from the outside atmosphere, allowing equilibration via the vapor phase.

Crystals of the GFR1 domain 3 were grown at +4°C in sitting drops over a reservoir solution of 50 raM MES, pH 6.5,... 

Alternatively, one can use sitting drop vapor diffusion where the drop is placed in a raised depression within the well. The sitting drop vapor diffusion experiment requires special plates or devices but set-up and retrieval of crystals is much simpler. Once the plate is prepared, it is then incubated at either 4 or 18°C for a period of time ranging from a few days to weeks. During incubation, each well of the plate is checked periodically under a microscope for the presence of crystals or precipitate. Conditions derived from the initial screen are usually further refined by adjusting pH, additives, and concentrations to grow suitable crystals for data collection and structnre analysis. Crystal seeding (Stura et al., 1992) can also be explored to improve the crystal size and quality. 

If the surface tension of the protein solution is rather low one should use the sitting drop method (b) which can be carried out in single drop or multiple drop vessels. Figure kindly supplied by J. Drenth, University of Groningen and reproduced with permission. 

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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