Department of Biological Chemistry, University of California Los Angeles, 615 Charles E Young Drive South, Los Angeles, CA 90095, USA; Department of Physiology, University of California Los Angeles, 615 Charles E Young Drive South, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles CA90095, USA.
Department of Biological Chemistry, University of California Los Angeles, 615 Charles E Young Drive South, Los Angeles, CA 90095, USA; Department of Physiology, University of California Los Angeles, 615 Charles E Young Drive South, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles CA90095, USA.
Structure. 2021 Jan 7;29(1):88-95.e2. doi: 10.1016/j.str.2020.09.003. Epub 2020 Oct 1.
A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy 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 approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.
一种高通量将配体浸泡到 TEM 网格上的蛋白质微晶体中的方法被提出。只需使用标准的 cryo-electron microscopy 玻璃化设备，就可以同时对网格上的每个晶体进行浸泡。该方法使用蛋白酶 K 微晶体和 5-氨基-2,4,6-三碘间苯二甲酸（I3C）魔术三角形进行了演示。使用聚焦离子束将浸泡的微晶体研磨至约 200nm 的厚度，并收集 MicroED 数据。确定了具有高占有率的四个配体的蛋白质的高分辨率结构。与通过 X 射线晶体学研究的类似处理的大得多的晶体相比，使用网格上的微晶体浸泡结合的配体数量及其占有率更高。这些结果表明，将配体浸泡到微晶体中可以有效地将配体吸收到蛋白质微晶体中。
