Practical techniques for crystallisation

Practical techniques for crystallisation have been reviewed [25,31,45,46]. Rapid screening of possible conditions are described by Carter and Carter [47] and Rayment [48], and McPherson [31] has documented nearly 200 crystallisation conditions for different proteins, providing a useful compendium which illustrates the diversity of methods employed. 

Variations on the vapour diffusion method have met with considerable success. A solution of the protein containing a salt concentration approximately 10% below that needed for precipitation is equilibrated by vapour diffusion with a larger volume of more concentrated salt solution which is only slightly below the concentration needed for precipitation. With non-volatile precipitants water distils out from the protein solution to the reservoir. With volatile solvents, distillation and equilibration will proceed in the opposite direction. The hanging drop version of this method allows numerous trials of different conditions with very little protein material. Plastic tissue culture plates (for example, with 24 cylindrical wells of 2 ml volume) may be used. The precipitant solution (1 ml volume) is placed in the wells. These are then sealed with a coverslip onto which a drop of protein solution (5-20 pi) has been placed and then inverted. A drop of light oil on the rim of the well makes for an air-tight seal. The method allows ready inspection of the drops without disturbing them. 

There have been several special cases where membrane proteins have been crystallised and high-resolution X-ray structures obtained. Where the protein is anchored in the membrane by a hydrophobic tail the protein may be released, for example, by proteases. The water-soluble component so released can be crystallised like a normal soluble globular protein. Outstanding examples of this approach have been cytochrome bi [49], influenza virus haemaglutinin [50] and influenza virus neuraminidase [51]. Alternatively, there have been a few examples of small proteins which can incorporate themselves into membranes and which are suflSciently homogeneous to be 

In these studies the protein was solubilised by the use of mild detergents and the protein in the micelle was the starting point for crystallisation using usual precipi-tants. The realisation that the detergent (its size and chemistry) played a direct role in crystallisation was an important step forward. The size and quality of the crystals were improved by addition of small polar organic molecules, which may act by improving packing of the micelles in the crystal lattice. 

Very recently, these methods have resulted in the first crystal structure determination by X-ray diffraction of an integral membrane protein, the photosynthetic reaction centre of Rps. viridis [265]. 

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