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International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 5.1, pp. 114-115
Section 5.1.2.2. Tools for crystal mounting
aThe Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111, USA |
In order to facilitate the process of mounting macromolecular crystals for X-ray diffraction experiments, it is necessary to have the appropriate tools for the task. Fig. 5.1.2.1![[link]](/graphics/greenarr.gif)
shows a collection of some useful tools for the mounting of crystals. These include a binocular microscope, tweezers (two types), thin glass capillaries, Pasteur pipettes, a heater, paper wicks, and a thumb pump. Other useful tools and supplies include surgical scissors, dental wax, latex tubing, light vacuum oil, a cryogenic mounting loop, Plasticine, mounting platforms, mounting pins, absorbent dental points and micropipettes with plastic tips. There are many other items that might be useful, and several variations are found in different laboratories. An important factor in the transfer of crystals from a holding solution to a capillary is that the experimenter needs to feel at ease with the process. The method that will be detailed here has evolved over time and has proved to be a relatively anxiety-free process. Other methods for crystal mounting may be found in the literature (Rayment, 1985; Sawyer & Turner, 1992; McRee, 1993). All of the methods outlined here and in the literature have the same goal, namely, the successful transfer of a macromolecular single crystal to a suitable mount for X-ray data collection.
![[Figure 5.1.2.1]](/figures/Fafig5o1o2o1thm.gif)
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Tools commonly used for mounting crystals. |
Perhaps the single most important piece of equipment for examining and mounting crystals is a binocular dissection microscope. This should have variable zoom capabilities, and there should be sufficient distance (e.g. 5–10 cm) between the objective lens of the microscope and the microscope stage to accommodate the necessary equipment and allow manipulation of the crystals and solutions. A magnification of between 10 and 40 times is probably best in practice. It is also important to ensure that the light source of the microscope is not so intense that it heats the microscope stage, thereby damaging the macromolecular crystals. If the microscope is fitted with crossed polarizers, the quality of the crystals can be assessed.
The capillaries used for crystal mounting are made of thin-walled glass. These capillaries range from 0.1 to 2.0 mm in diameter and have a stated wall thickness of 0.01 mm. In practice, however, the larger the diameter of the capillary, the thinner the glass wall. Therefore, handling of the larger-diameter capillaries is generally very difficult because they are so fragile. Capillaries made of fused quartz are also available, but are not recommended for general use because they produce a higher background with X-rays. Quartz capillaries are not as fragile as the thin-walled glass capillaries, however, and may be useful in experiments where the tensile strength of the capillary is important, for example, when a diffraction flow-cell experiment is planned (Petsko, 1985). In addition, small-diameter capillaries (produced in the laboratory by drawing out glass tubing or Pasteur pipettes) will be needed to aid in the removal of excess liquid around the crystal after the transfer from the crystallization dish to a capillary has been completed.
The thumb pump is a simple micropipetting device for transferring very small amounts of liquid in a highly controlled manner, making it an extremely useful tool for directly transferring protein crystals from solution to capillary, thus minimizing the chance of crystal damage. This simple device allows the experimenter to have much more control over volume transfers than any other device we have tried. The mechanism is simple and easy to operate. The device shown in the illustration can be held and manipulated with one hand. The capillary is held firmly in place and the pipetting action is controlled by a thumb wheel (part of the thumb pump), which affords a great deal of control over the volume of liquid being transferred with the crystal.
The heater illustrated here consists of a variable rheostat and a heating element. The latter is a short piece of Nichrome wire which has been coiled and attached to the rheostat via wires that run through a ball-point pen barrel. This permits a fine temperature control for melting dental wax and for controlling where the heat is applied.
References
Petsko, G. (1985). Flow cell construction and use. Methods Enzymol. 114, 141–146.Google Scholar
Rayment, I. (1985). Treatment and manipulation of crystals. Methods Enzymol. 114, 136–140.Google Scholar
Sawyer, L. & Turner, M. A. (1992). X-ray analysis. In Crystallization of nucleic acids and proteins. A practical approach, edited by A. Ducruix & R. Giegé, pp. 267–274. Oxford, New York, Tokyo: IRL Press.Google Scholar
