Gorrec Fabrice, Löwe Jan
Laboratory of Molecular Biology, Medical Research Council;
Laboratory of Molecular Biology, Medical Research Council.
J Vis Exp. 2018 Jan 24(131):55790. doi: 10.3791/55790.
When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.
当获得能够衍射X射线的高质量晶体时，晶体结构可以在接近原子分辨率的水平上得到解析。然而，蛋白质、DNA、RNA及其复合物的结晶条件是无法预测的。采用各种各样的条件是提高高质量衍射晶体产量的一种方法。英国剑桥医学研究理事会分子生物学实验室（MRC-LMB）开发了两种全自动系统，可通过纳升级液滴中的气相扩散针对1920种初始条件进行结晶筛选。还开发了半自动方案，通过改变试剂浓度、pH值或引入可能增强所得晶体性质的添加剂来优化条件。所有相应的方案将详细描述并简要讨论。这些方案共同作用，使得在多用户设施中进行方便且高效的大分子结晶成为可能，同时让用户能够控制实验的关键参数。
