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. 19.2, p. 424
Section 19.2.3.3. Other technical factors
aVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA |
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 . 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
). 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). 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
). 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
; Glaeser, 1992
; Booy & Pawley, 1993
). 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
). 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
). 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.
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