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 reliable technique that permits direct three-dimensional visualization of structural details at a level at which individual subunits in protein complexes or even single domains of proteins can be resolved. We describe in this paper a variation of the freeze-drying metal coating procedure that allows us to image with the STM the inner side of the metal 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 29 connector and the vesicle form of two-dimensional crystals of cytochrome oxidase from beef heart mitochondria. The images obtained have very good contrast and provide direct topographic information of the crystal surface, complementing structural information obtained previously with transmission electron microscopy. The resolution limit is imposed by the size (2-3 nm diameter) and corrugation of the metal grains used to prepare the replica and by the randomness of the metal shadowing.