Valpuesta J M, Henderson R, Frey T G
Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
J Mol Biol. 1990 Jul 5;214(1):237-51. doi: 10.1016/0022-2836(90)90158-I.
The structure of cytochrome oxidase from beef heart mitochondria has been analysed by cryo-electron microscopy of vesicle crystals of the space group p22(1)2(1), with cell dimensions a = 102 A, b = 123 A, gamma = 90 degrees. Several methods of specimen preparation were applied to the vesicular two-dimensional crystals in the electron microscope, to ensure that the structure was preserved to the maximum resolution. The two most informative density maps were from specimens embedded in ice and from negative staining in a 1:1 mixture of glucose and uranyl acetate. The three-dimensional structure of the ice-embedded molecule shows a single, well resolved, but convoluted density, which represents in size and shape one cytochrome oxidase dimer. At the bottom of the molecule, a substantial part of the protein is embedded in the lipid bilayer of the vesicle. The molecule then extends upwards, out of the bilayer, into the internal space within the vesicle. Here, the structure first passes through a region within the molecule containing a hollow cavity that lies roughly at the centre of mass of the dimer, and then branches into two well-resolved halves at some distance from the membrane. The negatively stained structure, in contrast, shows a stain-excluding region in the centre of the vesicle at the level of the cavity in the ice-embedded structure, but otherwise has a similar overall external shape. In addition, there is a small rotation of the whole molecule by approximately 25 degrees relative to the orientation of ice-embedded specimens. We interpret these differences to mean that the central cavity seen in the ice-embedded structure is too small to allow the stain to penetrate during the drying process and that the drying process causes the rotation. The structures described here are consistent with one another and allow an interpretation at higher resolution than from previous work.
通过对空间群为p22(1)2(1)的囊泡晶体进行冷冻电子显微镜分析，研究了牛心线粒体细胞色素氧化酶的结构，其晶胞参数为a = 102埃，b = 123埃，γ = 90度。在电子显微镜下，对囊泡二维晶体应用了几种标本制备方法，以确保结构能保留到最高分辨率。最具信息量的两张密度图分别来自嵌入冰中的标本和在葡萄糖与醋酸铀1:1混合物中进行负染色的标本。嵌入冰中的分子的三维结构显示出一个单一的、分辨率良好但呈卷曲状的密度，其大小和形状代表一个细胞色素氧化酶二聚体。在分子底部，相当一部分蛋白质嵌入囊泡的脂质双层中。然后分子向上延伸，离开双层，进入囊泡内部空间。在这里，结构首先穿过分子内一个大致位于二聚体质心处的中空腔区域，然后在距膜一定距离处分支为两个分辨率良好的部分。相比之下，负染色结构在嵌入冰的结构中腔的水平位置，在囊泡中心显示出一个排斥染色的区域，但总体外形相似。此外，整个分子相对于嵌入冰的标本的取向大约有25度的小旋转。我们将这些差异解释为，嵌入冰的结构中看到的中心腔太小，以至于在干燥过程中染色剂无法穿透，并且干燥过程导致了旋转。这里描述的结构相互一致，并且比以前的工作能在更高分辨率下进行解释。
