Two-Dimensional Crystallization of Biological Macromolecules - Molecules: Nucleation, Aggregation and Crystallization - page 107

in atomic models have been performed with liquid helium cryo-TEMs
(Fujiyoshi et al ., 1991). It has been demonstrated that electron diffraction
spots fade away slower when the specimen temperature is decreased
towards a few Kelvin, indicating reduced beam damage. Equally impor-
tant for improved information limits may be the stability of liquid helium
specimen stages.
Electron diffraction
It is a major advantage if the amplitude information of the Fourier com-
ponents can be obtained from electron diffraction data since the intensi-
ties will not be modulated by the influence of the objective lens like
amplitudes calculated from images. A recording using a CCD camera is
preferable. As compared to photographic film these have larger linear
dynamic range and initial evaluation of quality can be made online.
Images
If no approximate phase information is available such as from the struc-
ture of a very similar protein or from a different construct/complex of the
same protein, experimental phases have to be calculated from TEM
images of the 2D crystals. If images are recorded on film, a medium
which combines a large detection area with high resolution, the crystalline
areas have to be digitized using a high resolution scanner. Important con-
cepts to consider for the data collection are adjusting the dose in order to
avoid radiation damage without loss of too much contrast, nominal mag-
nification to adapt to the grain size of the film, the spot size of the scan-
ner, and the expected resolution.
Data processing
The basic programs for processing both electron diffraction data and
images are from the MRC package (Crowther et al ., 1996). This includes
indexing of diffraction patterns, extracting electron diffraction amplitudes,