Havelka W A, Henderson R, Oesterhelt D
Max-Planck Institut für Biochemie, Martinsried, Germany.
J Mol Biol. 1995 Apr 7;247(4):726-38. doi: 10.1006/jmbi.1995.0176.
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.
含有光驱动氯离子泵——嗜盐菌视紫红质的二维晶体斑块，似乎在过量生产嗜盐菌的菌株细胞膜中自发形成。已通过倾斜标本的电子冷冻显微镜分析了其三维结构（空间群p42(1)2，a = 102 Å）。图谱显示，嗜盐菌视紫红质（HR）具有七个跨膜螺旋的排列，类似于在相关质子泵细菌视紫红质（BR）中发现的排列。HR多肽骨架在膜中的方向相对于BR绕平面内的轴旋转了3度，并且排列在细胞质半通道的螺旋BC和FG之间的分子内空间在HR中似乎比在BR中略大，这对于氯离子通道来说是预期的。HR晶体太小，无法对倾斜标本进行电子衍射分析，因此傅里叶分量的幅度和相位均从图像中获得。这除了需要对散焦相位对比度传递函数进行校正外，还需要对图像幅度进行各向异性缩放。重新缩放数据的过程（在这种情况下大致相当于用-490的温度因子进行锐化）以补偿各种图像和晶体缺陷，在分析其他不存在同源结构先验知识且只能获得小晶体的结构时可能也会有用。
