CEA, CNRS, IBS, Université Grenoble Alpes.
CEA, CNRS, IBS, Université Grenoble Alpes; ELVESYS SAS.
J Vis Exp. 2021 Mar 13(169). doi: 10.3791/61685.
The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand. In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.
中子高分子晶体学（NMX）的应用正在迅速扩展，由于新的 NMX 光束线的建立以及结构精修软件的可用性增加，过去十年中确定了大多数结构。然而，目前用于 NMX 的中子源的强度明显低于 X 射线晶体学的等效源。尽管在该领域取得了进展，但对于中子衍射研究，总是需要更大的晶体，特别是研究越来越大的大分子和复合物的趋势。因此，需要进一步改进适用于生长更大晶体的方法和仪器，以便扩大 NMX 的使用。在这项工作中，我们介绍了我们实验室开发的合理策略和晶体生长台（OptiCrys），该台将显微镜安装的摄像机进行实时观察与结晶溶液的精确自动控制（例如沉淀剂浓度、pH 值、添加剂、温度）相结合。然后，我们展示了如何通过控制温度和化学成分来找到最佳的结晶条件，使用模型可溶性蛋白质进行演示。透彻了解结晶相图对于选择任何结晶实验的起始位置和动力学路径至关重要。我们展示了如何基于多维相图的知识来控制生成的晶体的大小和数量。
