Glaeser R M, Baldwin J, Ceska T A, Henderson R
Biophys J. 1986 Nov;50(5):913-20. doi: 10.1016/S0006-3495(86)83532-9.
High resolution electron diffraction data have been recorded for glucose-embedded purple membrane specimens in which bacteriorhodopsin (bR) has been trapped by cooling slowly to below--100 degrees C under continuous illumination. Thin films (OD approximately 0.7) of glucose-embedded membranes, prepared as a control, showed virtually 100% conversion to the M state, and stacks of such thin film specimens gave very similar x-ray diffraction patterns in the bR568 and the M412 state in most experiments. To be certain that any measured differences in diffraction intensity would be real, two independent sets of electron diffraction intensities were recorded for near-equatorial, i.e. (hkO), reflections. Little correlation was indeed observed between these two sets for delta F values at low resolution (15-5.0 A, 49 reflections), but the correlation coefficient is approximately 0.3 at high resolution (5.0-3.3 A, 218 reflections). Thus, while most of the measured difference is error, the mean delta F and the correlation coefficient can be used to estimate the smaller, true delta F due to structural changes occurring in the M state. The magnitude of this estimated true mean delta F is equal to what would be produced if approximately five to seven nonhydrogen atoms were moved to structurally uncorrelated (i.e., new) positions in the M state. Movements of a few amino acid side chains, and repositioning of atoms of the retinal group and the associated lysine side chain after trans-cis isomerization, are the most probable causes of the observed intensity changes in the M state. The difference Fourier map, calculated in projection at 3.5-A resolution, shows only very small peaks, the largest of which are confined, however, to the region of the protein.
已记录了葡萄糖包埋的紫膜标本的高分辨率电子衍射数据，其中细菌视紫红质（bR）在连续光照下缓慢冷却至低于 -100℃时被捕获。作为对照制备的葡萄糖包埋膜的薄膜（光密度约为0.7）显示几乎100%转化为M态，并且在大多数实验中，这种薄膜标本的堆叠在bR568和M412态下给出了非常相似的x射线衍射图谱。为确保任何测量到的衍射强度差异是真实的，针对近赤道反射，即（hkO）反射，记录了两组独立的电子衍射强度。对于低分辨率（15 - 5.0 Å，49个反射）下的ΔF值，这两组之间确实几乎没有相关性，但在高分辨率（5.0 - 3.3 Å，218个反射）下相关系数约为0.3。因此，虽然大部分测量差异是误差，但平均ΔF和相关系数可用于估计由于M态中发生的结构变化而产生的较小的真实ΔF。这个估计的真实平均ΔF的大小等于如果在M态中有大约五到七个非氢原子移动到结构不相关（即新的）位置时所产生的大小。少数氨基酸侧链的移动以及视黄醛基团和相关赖氨酸侧链的原子在反 - 顺异构化后的重新定位，是M态中观察到的强度变化的最可能原因。在3.5 Å分辨率下投影计算的差分傅里叶图仅显示非常小的峰，其中最大的峰局限于蛋白质区域。
