Section 3.4.1.4. Single crystals of biological macromolecules at ambient temperatures

Crystals of biological macromolecules are normally grown from an aqueous solution (see Subsection 3.1.1.2 ), and when growth is complete are in equilibrium with the mother liquor. Changes in this equilibrium may often result in crystal damage, so the most important aspect of crystal mounting in this case is to preserve the crystal in its state of hydration. This is most readily accomplished by sealing the crystal in a thin-walled quartz or glass capillary tube (King, 1954; Holmes & Blow, 1966). The crystal adheres to the inside of the tube by surface-tension effects through a small droplet of liquid, and a further pool of liquid at one end maintains the required degree of hydration. The general principles involved are well described by Rayment (1985). D'Aprile & Moretto (1975) have described two simple devices, a small electric heater for melting the wax used for sealing the capillary and a refrigerating microcell to prevent heat affecting the wet crystal, which are very useful for mounting wet single crystals in capillary tubes.

Alternatively, crystals can be grown directly within capillary tubes (Phillips, 1985) or microdialysis cells such as those described by Zeppezauer, Eklund & Zeppezauer (1968). A further mounting device particularly useful for enzymatic studies is the flow cell (Wyckoff et al., 1967), in which the specimen is immobilized while mother liquor, or buffer with substrates or inhibitors, is allowed to flow over the crystal. A useful account of this device is given by Petsko (1985). More recently, Edwards (1993) has described a yokeless flow cell, which uses a plastic cone fixed to a brass mounting pin with a wire harness to support the quartz capillary. Although the device was originally designed for Laue studies, its simplicity and practicality should make it useful for a wide range of diffraction experiments. Pickford, Garman, Jones & Stuart (1993) have designed a mounting cell that allows the humidity around a protein crystal to be varied in a controlled manner. This may be particularly useful for crystals where the solvent content is high and the molecular packing, and hence the diffraction intensities, highly dependent on the precise amount of solvent present.

The relatively short crystal lifetimes and large volumes of intensity data often dictate that crystals of biological macromolecules be mounted so that data collection can be accomplished in the most efficient manner, for example, with a symmetry axis parallel to the rotation axis of the collection device. Samples crystallizing in the form of thin plates that have to be aligned perpendicular to the capillary axis can be wedged using cotton lint fibres (Narayana, Weininger, Heuss & Argos, 1982), or mounted on a fibre plug (Przybylska, 1988).

One of the key problems in collecting diffraction data from wet crystals is movement of the specimen within the capillary, i.e. crystal slippage. Numerous ways have been suggested to surmount this problem, including flattening of the capillary surface, surrounding the crystal with a thin film of plastic (Rayment, Johnson & Suck, 1977) and supporting the crystal with fibre plugs in contact with the mother liquor.

Pressure cells. Tilton (1988) has described an attachment that can be used on conventional diffractometers for collecting X-ray data from biomolecular crystals under gas pressures up to 300 atm (30 MPa). The crystals are coated with mineral oil to minimize dehydration (see Subsection 3.4.1.5) and mounted in a quartz glass capillary between two layers of cotton fibres. These fibres give mechanical support to the specimen and protect it from shock during gas pressurization. No plugs of mother liquor or oil are used so that the gas flow is unimpeded. Kundrot & Richards (1986) describe an adaptation of the flow cell for hydrostatic pressure studies up to 0.2 GPa. More recently, Kroeger & Kundrot (1994) have described a gas cell that allows data sets at several partial pressures to be collected from the same crystal.