Crystallographic analysis of freeze-fractured three-dimensionally ordered specimens.
We describe here an original approach for solving the structure of three-dimensionally ordered specimens at low and medium resolutions. It combines freeze-fracture electron microscopy and quantitative image processing and has been first successfully applied to the crystallographic study of different lipid-containing cubic phases. The structure preservation during cryofixation is controlled by recording X-ray diffraction before and after freezing. Well frozen cubic phases show fracture planes which look like well defined cleavage planes of 3-D crystals. These fracture planes (domains) reveal a mosaic of 2D ordered sub-domains which are geometrically related to each other by simple crystallographic operations. The symmetry properties of the images mirror faithfully the symmetry of the space groups. The shifts and rotations observed between adjacent sub-domains are related to this symmetry. Different cubic phases display different fracture behavior, highly characteristic for a given space group. Interpretation of the averaged images of different domains in terms of molecular structure is done by the comparison of the averaged periodic motifs either with the corresponding sections of the electron density map (from X-ray diffraction data) or with the corresponding sections of a 3-D-space filling model. We show here that the same procedure may be applied to other three-dimensionally ordered specimens such as 3-D crystals of membrane proteins or of other proteins, including naturally occurring protein crystals of some secretory organelles. Finally, the same approach could also provide a powerful tool for the study of membrane protein crystallogenesis, particularly for the formation of 3-D crystals.
Centre de Génétique Moléculaire, CNRS, Gif-sur Yvette, France.
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Delacroix
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Gulik
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Gulik-Krzywicki
D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D003460:Crystallization; D003461:Crystallography / Q000379:methods*; D004075:Diglycerides / Q000737:chemistry; D005615:Freezing; D007091:Image Processing, Computer-Assisted; D008055:Lipids / Q000737:chemistry*; D008244:Lysophosphatidylcholines / Q000737:chemistry; D008823:Micelles; D010247:Paramecium / Q000737:chemistry / Q000502:physiology; D010713:Phosphatidylcholines / Q000737:chemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry; D012242:Rhodobacter sphaeroides / Q000737:chemistry; D014867:Water
D004075:Diglycerides; D008055:Lipids; D008244:Lysophosphatidylcholines; D008823:Micelles; D010713:Phosphatidylcholines; D045322:Photosynthetic Reaction Center Complex Proteins; D014867:Water; C017251:1,2-oleoylphosphatidylcholine; C013965:diolein