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Published on: April 11, 2021
Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking
Michael W Martynowycz1, Tamir Gonen1
1Department 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.
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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.
