Macromolecular Crystallography Research with Synchrotron Radiation

利用半胱氨酸和蛋氨酸残基的硫原子或所有蛋白质或RNA/DNA中固有的核酸的磷原子提供的非常小的异常衍射信号，可以成功地用于解决蛋白质的晶体结构。这种方法在一定程度上是由我们首创的，目前越来越受欢迎，并成为大分子晶体学实践中的常规技术。利用同步辐射从寡核苷酸和寡肽晶体中测量了0.6-0.8埃范围内的极高分辨率的衍射数据，改进的模型揭示了所研究分子的前所未有的结构细节，包括原子间sigma键的电子密度。在这种结构中观察到的非常精细的特征将有助于改进用于低分辨率蛋白质晶体结构常规细化的立体化学文库。我们参与了多个具有重要生物学意义的蛋白质结构合作项目，如荧光蛋白作为生物标志物，人类线粒体解旋酶，根瘤菌聚焦转移酶和植物s -腺苷-同型半胱氨酸水解酶。我们对大分子晶体的x射线辐射损伤效应进行了准确的评价，并确定了7 MGy的可接受吸收剂量极限。

Utilization of a very small anomalous diffraction signal provided by sulfur atoms of the cysteine and methionine residues or by phosphorus atoms of nucleic acids, intrinsically present in all proteins or RNA/DNA can be successfully used to solve crystal structures of proteins. This approach, in part pioneered by us, is currently gaining increasing popularity and becomes a routine technique in the practice of macromolecular crystallography. The diffraction data to extremely high resolution in the range of 0.6-0.8 angstrom were measured using synchrotron radiation from crystals of oligonucleotides and oligopeptides, and the refined models revealeed the unprecedented structural details of the investigated molecules including electron densities at the sigma bonds between atoms. The observed very fine features in this structures will serve to improve the stereochemical libraries used for routine refinement of protein crystal structures at lower resolution. We participated in several collaborative structural projects on various biologically important proteins, such as fluorescenty proteins used as biomarkers, human mitochindrial helicase, rhizobium fucosyltransferase and plant S-adenosyl-homocysteine hydrolase. We performed the accurate evaluation of the effects of X-ray radiation damage incurred in macromolecular crystals and established the limit of acceptable absorption dose at 7 MGy.