International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F. ch. 5.2, pp. 119-120   | 1 | 2 |

Section 5.2.6.6. Tomographic crystal-volume measurement

E. M. Westbrooka*

aMolecular Biology Consortium, Argonne, Illinois 60439, USA
Correspondence e-mail: westbrook@anl.gov

5.2.6.6. Tomographic crystal-volume measurement

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Recently, a new method for density measurement which is specific for protein crystals has been reported (Kiefersauer et al., 1996[link]). The crystal volume is calculated tomographically from a set of optical-shadow back projections of the crystal, with the crystal in many ([\gt\! 30]) orientations. This measurement is analogous to methods used in electron microscopy (Russ, 1990[link]). 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[link]). 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.

References

First citationHegerl, R. & Altbauer, A. (1982). The EM program system. Ultramicroscopy, 9, 109–116.Google Scholar
First citationKiefersauer, R., Stetefeld, J., Gomis-Rüth, F. X., Romão, M. J., Lottspeich, F. & Huber, R. (1996). Protein-crystal density by volume measurement and amino-acid analysis. J. Appl. Cryst. 29, 311–317.Google Scholar
First citationRuss, J. C. (1990). Computer-assisted microscopy: the measurement and analysis of images. New York: Plenum Press.Google Scholar








































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