Brink J, Van Breemen J F, Keegstra W, Van Bruggen E F
Biochemisch Laboratorium, Rijksuniversiteit Groningen, The Netherlands.
Ultramicroscopy. 1989 Jan-Feb;27(1):79-90. doi: 10.1016/0304-3991(89)90202-7.
We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.
我们研究了来自牛心线粒体NADH：泛醌氧化还原酶的二维晶体结构。对醋酸铀酰染色晶体的详细描述表明，它们由根据空间群P4212呈空间排列的片段组成[J. Brink，S. Hovmöller，C.I. Ragan，M.W.J. Cleeter，E.J. Boekema和E.F.J. van Bruggen，欧洲生物化学杂志166（1987）287]。为了获得更多关于晶体结构的结构信息并评估各种负染剂对结构保存和外观的影响，我们通过电子显微镜和图像分析检查了染色晶体。对于测试的几种染剂，即钼酸铵、醋酸铀酰、硝酸铀酰和硫酸铀酰，似乎都存在空间群P4212。使用磷钨酸和硅钨酸盐导致对称性降低到伪P4212或p4。使用钨酸钠导致分辨率大幅下降，最好只能达到3.8纳米，而在其他情况下可以证明分辨率为1.5至1.9纳米。晶格向量不受染剂影响；它们被确定为a = b = 14.9 +/- 0.25纳米，γ = 89.8度 +/- 0.6度。图像分析显示钼酸盐和铀酰化合物存在相似结构。在钨酸盐类型的染剂情况下观察到了差异。此外，分析揭示了晶胞中完全不存在四个直径为2.0纳米的小孔。无论染剂类型和支撑膜如何，都观察到了这种效应，并且只能归因于支撑膜的辉光放电处理。观察到的差异一定是由蛋白质、染剂和支撑膜之间相互作用的改变引起的。将对应分析和聚类算法应用于晶体的各种重建图像表明，它们可以分为几个簇。这些簇中的每一个平均仅对应一种染剂类型，而根据特定的铀酰化合物还观察到了进一步的划分。因此，这项研究表明，在相同的制备条件下，可以检测到个别染剂之间的细微差异。
