Lieberman Raquel L, Peek Mary E, Watkins J Derrick
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA.
Methods Mol Biol. 2013;955:475-93. doi: 10.1007/978-1-62703-176-9_25.
X-ray crystallography is a technique used to determine the atomic-detail structure of a biological macromolecule. The method relies on the ability to generate a three-dimensional crystal of a highly purified protein or nucleic acid for diffraction by X-rays. The extent of scattering of X-rays by the crystal determines the accuracy of the resulting structural model. Unlike electrons, X-rays cannot be refocused after they have been scattered by their target. Thus, calculations are needed to reconstruct the image of the macromolecule that builds the crystal lattice. Tremendous advances over the past 60 years in recombinant expression and purification, crystal growth methods and equipment, X-ray sources, computer processing power, programs, and graphics have taken X-ray crystallography from a highly specialized field to one increasingly accessible to researchers in the biomedical sciences. In this chapter, we review the major concepts of macromolecular X-ray crystallography, focusing mainly on techniques for crystallizing soluble and membrane proteins, and provide a protocol for the crystallization of lysozyme as a model for the crystallization of other proteins.
X射线晶体学是一种用于确定生物大分子原子细节结构的技术。该方法依赖于生成高度纯化的蛋白质或核酸的三维晶体以供X射线衍射的能力。晶体对X射线的散射程度决定了所得结构模型的准确性。与电子不同,X射线在被其目标散射后无法重新聚焦。因此,需要进行计算来重建构成晶格的大分子的图像。在过去60年里,重组表达与纯化、晶体生长方法与设备、X射线源、计算机处理能力、程序以及图形等方面取得了巨大进展,使得X射线晶体学从一个高度专业化的领域发展成为生物医学领域研究人员越来越容易涉足的领域。在本章中,我们将回顾大分子X射线晶体学的主要概念,主要聚焦于可溶性和膜蛋白的结晶技术,并提供溶菌酶结晶的实验方案,作为其他蛋白质结晶的模型。