Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing

Abstract

We have compared the appearance and preservation of molecular and supramolecular structures in preparations that were dried in vacuo at room temperature or freeze-dried. Fibrinogen and brain spectrin molecules appear similar in both types of preparation provided that drying at room temperature is performed in the presence of glycerol, which results in an even and reproducible distribution of such molecules (Shotton et al., 1979, J. Mol. Biol. 131, 303-329; Fowler and Erickson, 1979, J. Mol. Biol. 134, 241-249). In the case of crystalline actin sheets, actin filaments, and keratin filaments, freeze-drying preserves structural details that are often completely lost during drying at room temperature, whether or not glycerol is used. On the other hand, keratin filaments prepared by drying in the presence of glycerol display a beaded axial repeat that is probably due to "glycerol decoration." We conclude that although freeze-drying has no clear advantage over glycerol spraying/vacuum-drying in the case of single extended molecules, it may provide insight into the multiple effects of glycerol in specimen preparation. In the case of supramolecular assemblies such as filaments or crystalline sheets, freeze-drying preserves significantly more substructure and surface detail. The loss of such detail during drying at room temperature, probably through collapse phenomena such as distortion and flattening, cannot be prevented by glycerol.