A method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

中文翻译：

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通过并网浸泡有效地将高通量配体掺入蛋白质微晶中

提出了一种将配体浸入TEM网格上的蛋白质微晶中的方法。只能使用标准cryoEM玻璃化设备同时浸泡网格上的每个晶体。使用浸泡有5-氨基-2,4,6-三碘间苯二甲酸（I3C）魔三角的蛋白酶K微晶证明了该方法。使用聚焦离子束将浸透的微晶研磨至200nm的厚度，并收集微晶电子衍射（MicroED）数据。确定了具有四个高配体的蛋白质的高分辨率结构。与通过X射线晶体学研究的更大的晶体相比，在MicroED中结合的配体数量和它们的占有率都更高。