Molecular orientation of bacteriorhodopsin within the purple membrane of Halobacterium halobium

The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.