Section 19.2.3.1. Specimen preparation

An electron microscope column is kept at a pressure of < 10⁻⁶ Torr (1 Torr = 133.322 Pa). Because a thin protein crystal loses its crystallinity if dried in a vacuum, its hydration can be maintained by embedding it in a thin layer of vitreous ice, glucose, or other small sugar derivatives (Unwin & Henderson, 1975; Dubochet et al., 1988). The effectiveness of these preservation methods is evidenced by the high-resolution diffraction orders (out to at least 3 Å) from properly embedded protein crystals (Fig. 19.2.3.1). Since the high-resolution reflections come mostly from the protein, their diffraction intensities are largely independent of the embedding medium. However, the low-resolution diffraction intensities can be affected by the embedding medium because different media have different scattering densities relative to the protein. For any new crystal, any of the embedding media mentioned above can be used for high-resolution structural studies.

Figure 19.2.3.1| top | pdf | Electron diffraction pattern of trehalose-embedded bacteriorhodopsin, with Bragg reflections extending to 2.5 Å. The unit-cell parameter of this 45 Å-thick membrane protein crystal is 62.5 × 62.5 Å arranged in a p3 two-dimensional space group. The raw diffraction pattern was recorded on a Gatan 2k × 2k CCD camera with 300 kV electrons in a JEOL 3000 electron cryomicroscope equipped with a field emission gun and a liquid-helium (4 K) cryoholder. The pattern displayed has been contrast-enhanced using radial background subtraction. A central beam stop was used to prevent saturation of the detector but has blocked off some reflections. for the Friedel-symmetry-related reflections (about 290 pairs) was computed to be about 5%. (Courtesy of Drs Yifan Cheng and Yoshinori Fujiyoshi at Kyoto University.)