REVERSED METAL REPLICAS OF FREEZE-DRIED PROTEINS TO BE VISUALIZED WITH THE SCANNING TUNNELING MICROSCOPE

Scanning tunneling microscopy of metal-coated specimens has become a r eliable technique that permits direct three-dimensional visualization of structural details at a level at which individual subunits in prote in complexes or even single domains of proteins can be resolved. We de scribe in this paper a variation of the freeze-drying metal coating pr ocedure that allows us to image with the STM the inner side of the met al replica, previously in contact with the protein molecules, We have tested this new approach with two different well characterized protein systems: freeze-dried two-dimensional crystals of bacteriophage Phi 2 9 connector and the vesicle form of two-dimensional crystals of cytoch rome oxidase from beef heart mitochondria. The images obtained have ve ry good contrast and provide direct topographic information of the cry stal surface, complementing structural information obtained previously with transmission electron microscopy, The resolution limit is impose d by the size (2-3 nm diameter) and corrugation of the metal grains us ed to prepare the replica and by the randomness of the metal shadowing .