School of Biological Sciences, The University of Adelaide.
School of Biological Sciences, The University of Adelaide; Institute of Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide.
J Vis Exp. 2021 Jan 16(167). doi: 10.3791/61894.
Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.
使用 X 射线晶体学阐明蛋白质结构需要高质量的衍射晶体和衍射相问题的计算解决方案。缺乏合适同源模型的新型结构通常通过用重原子衍生化来提供实验相信息。本方案通过将随机微种基质筛选与用重原子分子 I3C（5-氨基-2,4,6-三碘异苯二甲酸）衍生化相结合，有效地生成了衍生化的蛋白质晶体。通过将 I3C 掺入晶格中，可以使用单波长反常散射（SAD）相测定有效地解决衍射相问题。I3C 中碘原子的等边三角形排列允许快速验证正确的反常亚结构。对于使用基于晶体学的技术解决大分子结构并对实验相感兴趣的结构生物学家来说，本方案将非常有用。
