Vapor diffusion method

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 ... 

B. Zheng, J.D. Tice, L.S. Roach, and R.F. Ismagilov A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with on-Chip X-Ray Diffraction. Angew. Chem. Int. Ed. 43, 2508 (2004). 

Fig. 11 Wet thickness (H) of PAAm in water as a function of the PAAm graft density for samples prepared by surface-initiated ATRP on substrates with gradient of initiator density. The initiator was immobilized by the vapor-diffusion method using mixtures of l-trichlorosilyl-2-(fn/p-chloromethyl phenyl)ethan n-octyl trichlorosilane (w/w) 1 1 (squares), 1 2 (circles), and 1 5 (triangles). The inset shows a cartoon illustrating the polymer behavior. Reproduced with permission from [134] (Copyright 2003 American Chemical Society)...

Superconducting films of C60 compounds with alkali metals (M3C6o) were first prepared at AT T Bell Laboratories by means of this vapor diffusion method (Haddon and others 1991). Meanwhile, pure K3C60 can be prepared simply by mixing powdered potassium metal and C6o in toluene (Wang et al. 1991). 

Miller, T. Y., He, X.M., Carter, D.C., A comparison between protein crystals grown with vapor diffusion methods in microgravity and protein crystals using a gel liquid liquid diffusion ground-based method. J. Cryst. Growth 1992, 122 (1-4), 306-309. 

The most unpredictable process in X-ray structure determination is the crystallization of the candidate protein into a form suitable for X-ray diffraction. Each protein requires a unique set of conditions to form crystals. Typically 100 mg of highly purified protein is required to determine the conditions that result in usable crystals of 0.1 to 0.3 mm size, although a size of 0.3 to 0.8 mm is preferred. The occurrence of crystals and the rate of crystallization are influenced by many factors such as protein purity, the solvent, concentration of added precipitants, pH, temperature, and the presence of ions and cofactors. The protein solution at a concentration of typically 5 to 20 mg/ml is allowed to slowly reach supersaturation by the removal of or by changing the composition of the solvent by liquid-liquid diffusion or vapor diffusion methods. Microscale methods have been developed to explore several crystallization conditions simultaneously using minimum amounts of the purified protein sample. Recently, use of the zero gravity atmosphere in space has been explored as a means of facilitating crystallization (Eisenberg and Hill, 1990 Branden and Tooze, 1991 Tomasselli et al, 1991). 

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. 

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

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. 

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. 

In addition to the vapor diffusion method described previously, other techniques such as the batch and micro-batch methods, bulk and micro dialysis, free interface diffusion, liquid bridge, and concentration dialysis have also been developed to produce crystals for x-ray diffraction analysis (see McPherson, 1982 and McPherson, 1999). 

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.

B. Zheng, J. D. Tice, L. S. Roach, and R. F. Ismagilov, A droplet-based, composite PDMS/glass capillary microfluidic system for evaluating protein crystallization conditions by microbatch and vapor-diffusion methods with on-chip X-ray diffraction, Angewandte Chemie-lntemational Edition, vol. 43, no. 19, pp. 2508-2511, 2004. 

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