Ceska T A, Henderson R
MRC Laboratory of Molecular Biology, Cambridge, England.
J Mol Biol. 1990 Jun 5;213(3):539-60. doi: 10.1016/S0022-2836(05)80214-1.
Progress in the structure determination of bacteriorhodopsin, the protein component of purple membrane from Halobacterium halobium has been limited by the lack of three-dimensional phase information between 6 and 3 A resolution. By analogy with X-ray methods, it is possible that heavy-atom labelling of the membrane crystal may provide heavy-atom derivatives that can be used for phasing by the multiple isomorphous replacement method. This paper describes the screening of heavy-atom compounds as potential derivatives, and the evaluation of the data collected from these heavy-atom-labelled membranes. Improvements in the methods for collecting electron diffraction data and analysing and merging the data are presented. Diffraction patterns of purple membrane samples were taken at -120 degrees C to minimize radiation damage. About 30 heavy-atom compounds were tested for use as potential derivatives. The diffraction patterns from labelled membranes were analysed by examining 6.5 A difference Fourier maps. Two heavy-atom compounds were selected for three-dimensional data collection at 3 A resolution. In addition, a full set of native data at -120 degrees C was collected to 2.7 A resolution. The intensity merging, heavy-atom derivative evaluation, heavy-atom refinement and the calculation of phases are presented. Phases are compared to those determined by electron microscope imaging, and limitations of the method are discussed. It is concluded that, with the present accuracy of data collection and the present magnitude of delta F/F available for the derivatives, the phasing power is too small. The phases that are obtained are not sufficiently accurate to provide a reliably interpretable map. It may be possible, however, to use the heavy-atom derivative data in difference Fourier calculations in which the presence of a peak would confirm the phases calculated from a model or obtained by electron microscope imaging.
嗜盐菌紫膜蛋白细菌视紫红质的结构测定进展一直受到6至3埃分辨率之间三维相位信息缺乏的限制。通过与X射线方法类比，膜晶体的重原子标记有可能提供可用于多重同晶置换法相位测定的重原子衍生物。本文描述了作为潜在衍生物的重原子化合物的筛选，以及对从这些重原子标记膜收集的数据的评估。文中介绍了收集电子衍射数据以及分析和合并数据方法的改进。在-120℃下获取紫膜样品的衍射图样以尽量减少辐射损伤。测试了约30种重原子化合物作为潜在衍生物。通过检查6.5埃的差值傅里叶图来分析标记膜的衍射图样。选择了两种重原子化合物用于3埃分辨率的三维数据收集。此外，还在-120℃下收集了一套完整的天然数据，分辨率达到2.7埃。文中展示了强度合并、重原子衍生物评估、重原子精修以及相位计算。将相位与通过电子显微镜成像确定的相位进行了比较，并讨论了该方法的局限性。得出的结论是，就目前的数据收集精度和衍生物可获得的ΔF/F量级而言，相位测定能力太小。所获得的相位不够精确，无法提供可靠可解释的图谱。然而，有可能在差值傅里叶计算中使用重原子衍生物数据，其中峰值的出现将确认从模型计算或通过电子显微镜成像获得的相位。
