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.
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 70 K
Image recording
Average exposure time: 1 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 240 / Num. of grids imaged: 1 / Num. of real images: 240
Image scans
Sampling size: 28 µm / Width: 4096 / Height: 4096
EM diffraction
Camera length: 1900 mm / Tilt angle list: -30,30
EM diffraction shell
Resolution: 43.32→1.78 Å / Fourier space coverage: 94.52 % / Multiplicity: 4.9 / Num. of structure factors: 10458 / Phase residual: 18 °
EM diffraction stats
Fourier space coverage: 94.52 % / High resolution: 1.78 Å / Num. of intensities measured: 109326 / Num. of structure factors: 10458 / Phase error: 18 ° / Phase residual: 18 ° / Phase error rejection criteria: 0 / Rmerge: 0.26 / Rsym: 0.13
Reflection
Biso Wilson estimate: 14.37 Å2
-
Processing
Software
Name
Version
Classification
NB
phenix.refine
1.17.1_3660
refinement
PHENIX
1.17.1_3660
refinement
EM 3D crystal entity
∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.55 Å / B: 67.55 Å / C: 102.77 Å / Space group name: 96 / Space group num: 96
CTF correction
Type: NONE
3D reconstruction
Resolution: 1.78 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model building
B value: 13.51 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: maximum liklihood
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