Design and implementation of suspended drop crystallization.
Design and implementation of suspended drop crystallization.
复制标题
作者：
在这项研究中，设计并3D打印了一种用于晶体生长的装置，该装置消除了电子显微镜中的择优取向问题以及在微晶电子衍射之前处理晶体滴的需要。该装置上的酶蛋白酶K进行了测试，并确定其结构的MicroED使用这种方法。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。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，允许蒸汽从液滴的两侧扩散。网格上方和下方的紫外线透明窗口可以通过光学、紫外线或荧光显微镜监测晶体生长。一旦晶体形成，网格可以被移除并直接用于X射线晶体学或微晶电子衍射（MicroED），而不必操纵晶体。为了证明这种方法的有效性，生长酶蛋白酶K的晶体，并通过聚焦离子束/扫描电子显微镜研磨后的MicroED确定其结构，以使样品足够薄用于cryoEM。悬浮液滴结晶克服了许多与样品制备相关的挑战，为嵌入粘性介质中的晶体提供了替代工作流程，对机械应力敏感和/或在电子显微镜网格上受到优先取向。
In this study an apparatus for crystal growth which eliminates the preferred orientation problem in electron microscopy and the need for handling of crystal drops prior to microcrystal electron diffraction was designed and 3D-printed. The apparatus was tested on the enzyme proteinase K and its structure was determined by MicroED using this approach. 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.