PDF(Pubmed)
Abstract：
In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.
摘要：
在这项工作中,一种新的晶体生长方法称为悬浮滴结晶已经开发出来。与传统方法不同，该技术涉及直接在电子显微镜网格上混合蛋白质和沉淀剂，而无需任何额外的支撑层。然后将网格悬挂在内部设计的结晶室内，允许从液滴的两侧发生蒸汽扩散。网格上方和下方的紫外线透明窗口可以通过光监测晶体生长，紫外或荧光显微镜。一旦晶体形成，可以去除网格并直接用于X射线晶体学或微晶体电子衍射(MicroED)，而不必操纵晶体。为了证明这种方法的有效性，生长蛋白酶K的晶体，并在聚焦离子束/扫描电子显微镜研磨后通过MicroED确定其结构，以使样品足够薄以进行冷冻EM。悬浮液滴结晶克服了与样品制备相关的许多挑战，为嵌入粘性介质中的晶体提供替代工作流程，对机械应力敏感和/或在电子显微镜网格上受到优选的取向。
