Molecular structure determination of crystalline specimens in frozen aqueous solutions.

Images were obtained of eukaryotic ribosome crystals, bladder membranes and gap junctions preserved in frozen aqueous solutions under conditions where either amorphous or crystalline ice is formed. Evaluation of these images by optical diffraction showed that specimens containing the largest open spaces were sensitive to ice crystal damage during freezing, whereas those containing the smallest open spaces were not. Projection maps were calculated from the images and compared to maps obtained from the same specimens at the same resolution in negative stain. Significant differences were apparent between each pair of maps. These were attributed to details being revealed of the RNA and protein (ribosomes) or the complete protein (membranes) when using frozen solutions, compared to just the hydrophilic surfaces when using stain. Thus the freezing method appears to provide the most complete and accurate descriptions of these structures.