National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41GM136508
United States
Citation
Journal: Structure / Year: 2021 Title: Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking. Authors: Michael W Martynowycz / Tamir Gonen / Abstract: 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 ...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.
History
Deposition
Aug 16, 2020
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Header (metadata) release
Oct 14, 2020
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Map release
Oct 14, 2020
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Update
Jan 20, 2021
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Current status
Jan 20, 2021
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.78 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Crystal parameters
Unit cell - A: 67.55 Å / Unit cell - B: 67.55 Å / Unit cell - C: 102.77 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: 96
Crystallography statistics
Number intensities measured: 109326 / Number structure factors: 10458 / Fourier space coverage: 94.52 / R sym: 0.13 / R merge: 0.26 / Overall phase error: 18 / Overall phase residual: 18 / Phase error rejection criteria: 0 / High resolution: 1.78 Å / Shell - Shell ID: 1 / Shell - High resolution: 43.32 Å / Shell - Low resolution: 1.78 Å / Shell - Number structure factors: 10458 / Shell - Phase residual: 18 / Shell - Fourier space coverage: 94.52 / Shell - Multiplicity: 4.9
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