Combined use of freeze-fracture electron microscopy and X-ray diffraction for the structure determination of three-dimensionally ordered specimens

The cubic phases of lipid-water systems have been studied by freeze-fracture electron microscopy. The preservation of the sample structure following cryofixation was verified by low temperature X-ray diffraction. Different types of fracture planes were identified; all display highly ordered two-dimensional domains, each subdivided into sub-domains related to each other by displacements and rotations related to the symmetry of the space group. The images were filtered using cross-correlation averaging techniques and the filtered images were compared to the corresponding planar sections of the electron density maps. Several conclusions were drawn: 1) when properly cryofixed, as assessed by low temperature X-ray diffraction, the structure of the sample was well preserved in the replicas; 2) the symmetry of the space group was faithfully reflected in the electron microscope images; 3) the crystallographic orientations of the most frequently identified'fracture planes coincided with those of the most intense X-ray reflections indicating that the fracture propagates, preferentially, in regions where the electron density variations are the largest; 4) when different structural models are compatible with X-ray diffraction data, it is possible to determine the correct model by comparing the filtered images with sections of the corresponding electron density maps; and 5) this approach constitutes a new and powerful tool of general interest for the low resolution study of three-dimensionally ordered specimens.