Section 5.2.6.6. Tomographic crystal-volume measurement

Recently, a new method for density measurement which is specific for protein crystals has been reported (Kiefersauer et al., 1996). The crystal volume is calculated tomographically from a set of optical-shadow back projections of the crystal, with the crystal in many () orientations. This measurement is analogous to methods used in electron microscopy (Russ, 1990). The crystal is mounted on a thin fibre which is in turn mounted on a goniostat capable of positioning it in many angular orientations. The crystal must remain bathed in a humidity-regulated air stream to avoid drying. The uncertainty of the volume measurement improves asymptotically as the number of orientations increases (estimated to be 10–15%). The images are captured by a digital charge-coupled device camera, transferred to a computer and processed with the program package EM (Hegerl & Altbauer, 1982). This same crystal must then be recovered and subjected to quantitative amino-acid analysis (the authors used a Beckman 6300 amino-acid analyser). With a lower limit of 100 pmol for each amino acid, the uncertainty of this measurement was estimated to be 10–20% for typical protein crystals. The method appears to work for crystals with volumes ranging between 4–50 nl. Errors in the determined values of n ranged from 4–30%.

Implementation of the method requires complex equipment and considerable commitment (in terms of hardware and software) by the research laboratory. The accuracy of the method is sufficient to determine n unambiguously in many cases, but it is not as high as can be obtained with gradient-tube or flotation methods if care is taken. The method has the virtue that once established in a research laboratory, it might lend itself to a considerable degree of automation, thereby reducing the activation barrier to measuring crystal densities for members of the research group.