Imaging a Protein α-Helix by Dark Field Electron Microscopy
Elucidation of the fine structure of a protein, beyond its gross conformation, requires images of better than 10 Å resolution. In order to achieve such a resolution the normal techniques of staining the molecule must be avoided by substituting a technique such as dark field electron microscopy. However, to form a dark field image of such high resolution requires an exposure of approximately 1500 e/Å2. Exposures of this magnitude are so high that serious doubt has been expressed regarding the extent to which an image formed under these circumstances is an accurate representation of the molecule. Images of myokinase, protamine, glucagon, secretin, and ACTH indicate that reproducible 5-10 Å detail is present in the micrographs. Furthermore, for bacitrin, valinomycin, and the growth stimulators LMW-CSA N and B, biologically relevant information of 3-5 Å is present in averages prepared from a collection of images or even in single molecular images. Visual recognition has been used as a criterion to assess radiation damage in images of isolated molecules of protamine.