Two-dimensional crystalline patches containing the light-driven chloride pump, halorhodopsin, appear to form spontaneously in the cell membrane of an overproducing strain of Halobacterium. The three-dimensional structure (space group p42(1)2, a = 102 A) has been analysed by electron cryo-microscopy of tilted specimens. The map shows that halorhodopsin (HR) has an arrangement of seven transmembrane helices similar to that found in the related proton pump bacteriohodopsin (BR). The orientation of the polypeptide framework of HR in the membrane is rotated by 3 degrees relative to BR about an axis in the plane and the intramolecular space between the helices BC FG, which line the cytoplasmic 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 amplitudes and the phases of the Fourier components were obtained from images. This required anisotropic scaling of the image amplitudes in addition to correction for the defocus phase contrast transfer function. The procedure of rescaling the data (in this case roughly equivalent to sharpening with a temperature factor of-490) to compensate for a variety of image and crystal defects may also prove useful in the analysis of other structures for which no prior knowledge of a homologous structure exists and for which only small crystals can be obtained.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Havelka WA, Henderson R, Oesterhelt D

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