3-DIMENSIONAL STRUCTURE OF HALORHODOPSIN AT 7-ANGSTROM RESOLUTION

Two-dimensional crystalline patches containing the light-driven chlori de pump, halorhodopsin, appear to form spontaneously in the cell membr ane of an overproducing strain of Halobacterium. The three-dimensional structure (space group p42(1)2, a=102 Angstrom) has been analysed by electron cryo-microscopy of tilted specimens. The map shows that halor hodopsin (HR) has an arrangement of seven transmembrane helices simila r to that found in the related proton pump bacteriohodopsin (BR). The orientation of the polypeptide framework of HR in the membrane is rota ted by 3 degrees relative to BR about an axis in the plane and the int ramolecular space between the helices BC FG, which line the cytoplasmi c half channel, appears slightly larger in HR than in BR, as would be expected for a chloride channel. The crystals of HR were too small for electron diffraction analysis of tilted specimens, so both the amplit udes and the phases of the Fourier components were obtained from image s. This required anisotropic scaling of the image amplitudes in additi on to correction for the defocus phase contrast transfer function. The procedure of rescaling the data (in this case roughly equivalent to s harpening with a temperature factor of -490) to compensate for a varie ty of image and crystal defects may also prove useful in the analysis of other structures for which no prior knowledge of a homologous struc ture exists and for which only small crystals can be obtained.