Section 19.2.3.2. Radiation damage

All protein crystals are prone to radiation damage caused by inelastically scattered electrons (Glaeser, 1971). This physical process is easily seen in the fading of electron diffraction intensities of a protein crystal as the accumulated doses increase. The consequence of damage is a preferential loss of the high-resolution information. Radiation damage is a dose-dependent process and cannot be reduced by adjusting the dose rate (flux) of the irradiating electrons. The strategy used to minimize the damage is to record the diffraction or image data from a specimen area that has not been previously exposed to electrons for purposes of focusing or other adjustments (Unwin & Henderson, 1975). This is called a minimal or low-dose procedure. In addition, keeping a specimen at low temperature (<113 K) allows it to tolerate a higher radiation dose (by a factor of about 4 to 6) before reaching the same extent of damage as at room temperature (Hayward & Glaeser, 1979). It has been shown that damage reduction is minimal below liquid-nitrogen temperature (Chiu et al., 1981). However, there have been some impressive results using the electron cryomicroscope to study membrane protein crystals kept at liquid-helium temperature (4 K) (Kühlbrandt et al., 1994; Kimura et al., 1997; Miyazawa et al., 1999).