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用高分辨电镜方法研究不染色的蛋白质分子结构的主要困难有二:样品对电子损伤的高敏感性;样品在真空中三维结构的改变。我们采用性质与水相似而又不挥发的葡萄糖取代水介质,以防止真空损伤。用低剂量(<1e/A~2)电镜技术防止幅射损伤。然而由于样品固有低反差和低剂量成象,象的S/N非常低,以致不能直接观察。但如果样品是严格周期结构并具有足够多的分子或单胞,则重构分子或单胞所需的信息就可从计算机中萃取出来。为了重构需要测定Fourier函数的相位和振幅。两者分别由显微象的Fourier分析和电子衍射花样的测定而被确定。本文讨论测定Catalase和B.Subtilis α-Amylase晶体结构的实验方法。
There are two major difficulties in using high-resolution electron microscopy to study non-stained protein molecular structures: high sensitivity of the sample to electron damage; and changes in the three-dimensional structure of the sample in a vacuum. We replaced aqueous media with dextrose similar to water but not volatile to prevent vacuum damage. Low dose (<1e / A ~ 2) electron microscopy to prevent radiation damage. However, the S / N of the image is so low that it can not be directly observed because of the inherently low contrast and low dose imaging of the sample. But if the sample is strictly periodic and has enough molecules or cells, the information needed to reconstruct the molecules or cells can be extracted from the computer. For reconstruction it is necessary to determine the phase and amplitude of the Fourier function. Both were determined by the Fourier analysis of microscopic images and the determination of electron diffraction patterns. This article discusses the experimental methods for determining the crystal structure of Catalase and B. Subtilis α-Amylase.