Freeze-fracture electron microscopic and low temperature x-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure.

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL2) structure. Only when the LDL2 solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL2 X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL2 X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freeze-etching electron microscopy of the slowly cryofixed LDL2 showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL2 lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.