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. 19.2, p. 424   | 1 | 2 |

Section 19.2.3.3. Other technical factors

W. Chiua*

aVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
Correspondence e-mail: wah@bcm.tmc.edu

19.2.3.3. Other technical factors

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In order to record a three-dimensional data set, the crystals have to be tilted to different angles with respect to the direction of the electron beam. In a typical electron microscope, the highest angle to which the specimen stage can be tilted is about [\pm 60^{\circ}]. Consequently, there is a missing set of data beyond the highest tilt angle, which corresponds to no more than 15% of the entire three-dimensional volume. Because of the radiation damage, a single diffraction pattern or a single image per crystal is usually recorded (Henderson & Unwin, 1975[link]). The quality of a crystal is easily judged by its electron diffraction pattern as captured from a CCD camera during data collection. Evaluating the ultimate quality of images, however, takes more time and requires extensive computational analysis.

There are two major technical problems that often limit the data quality, even though a crystal is highly ordered (Henderson & Glaeser, 1985[link]). One is the flatness of the crystal, and the other is the beam-induced movement or charging of the crystal. The effects of both problems become more prominent when the crystals are tilted to high angles. These effects tend to blur the diffraction spots, resulting in loss of high-resolution data (Brink, Sherman et al., 1998[link]). There are many ways to overcome these technical handicaps. For instance, the type of microscope grid chosen or the method of making the carbon support film is critical for reducing the wrinkling of the crystals (Butt et al., 1991[link]; Glaeser, 1992[link]; Booy & Pawley, 1993[link]). The use of a carbon film, which is a good conducting material, to support the protein crystal appears to reduce specimen charging (Brink, Gross et al., 1998[link]). It has been suggested that using a gold-plated objective aperture is effective in reducing specimen charging by generating a stream of secondary electrons to neutralize the positive charges that have built up on the specimen, which thus acts like an aberration-inducing electrostatic lens. Empirically, irradiating the microscope grid before depositing the specimen also reduces the charging (Miyazawa et al., 1999[link]). All these technical problems that can hamper progress in the completion of the structure determination have gradually been identified and resolved. However, more convenient and more robust experimental procedures for reducing these effects further are desirable in order to enhance the efficiency of data collection.

References

First citation Booy, F. P. & Pawley, J. B. (1993). Cryo-crinkling: what happens to carbon films on copper grids at low temperature. Ultramicroscopy, 48, 273–280.Google Scholar
First citation Brink, J., Gross, H., Tittmann, P., Sherman, M. B. & Chiu, W. (1998). Reduction of charging in protein electron cryomicroscopy. J. Microsc. 191, 67–73.Google Scholar
First citation Brink, J., Sherman, M. B., Berriman, J. & Chiu, W. (1998). Evaluation of charging on macromolecules in electron cryomicroscopy. Ultramicroscopy, 72, 41–52.Google Scholar
First citation Butt, H. J., Wang, D. N., Hansma, P. K. & Kühlbrandt, W. (1991). Effect of surface roughness of carbon support films on high-resolution electron diffraction of two-dimensional protein crystals. Ultramicroscopy, 36, 307–318.Google Scholar
First citation Glaeser, R. M. (1992). Specimen flatness of thin crystalline arrays: influence of the substrate. Ultramicroscopy, 46, 33–43.Google Scholar
First citation Henderson, R. & Glaeser, R. M. (1985). Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals. Ultramicroscopy, 16, 139–150.Google Scholar
First citation Henderson, R. & Unwin, P. N. (1975). Three-dimensional model of purple membrane obtained by electron microscopy. Nature (London), 257, 28–32.Google Scholar
First citation Miyazawa, A., Fujiyoshi, Y., Stowell, M. & Unwin, N. (1999). Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall. J. Mol. Biol. 288, 765–786.Google Scholar








































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