Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size
Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size
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DOI：
10.1016/j.str.2018.12.003
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发表时间：
2019-03-05
期刊：
影响因子：
5.7
通讯作者：
Gonen, Tamir
中科院分区：
文献类型：
--
作者：
Martynowycz, Michael W.;Zhao, Wei;Gonen, Tamir
微晶电子衍射（MicroED）允许从纳米晶体的大分子结构的解决方案。为了产生用于MicroED数据收集的合适尺寸的晶体，样品制备通常涉及超声处理或从结晶液滴中移取晶体浆料。所得的晶体碎片是易碎的，并且可以从它们获得的数据的质量对随后用于冷冻电子显微镜的样品制备是敏感的，因为水-空气界面中的相互作用可以在印迹期间损坏晶体。在这里，我们展示了使用聚焦离子束来产生大分子蛋白质晶体的薄片，用于连续旋转MicroED，其具有理想的厚度，易于定位，并且不需要印迹优化。通过这种方式，在数据收集之前，几乎任何尺寸的晶体都可以被舀取并研磨至所需的尺寸，从而简化了MicroED样品制备的方法。
Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.