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Microcrystal Electron Diffraction of Small Molecules

Published: March 15th, 2021



1Howard Hughes Medical Institute, University of California Los Angeles, 2Department of Biological Chemistry, University of California Los Angeles, 3Department of Physiology, University of California Los Angeles

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) method for determining atomic resolution structures from sub-micrometer sized crystals1,2. Crystals are applied to standard transmission electron microscope (TEM) grids and frozen by either plunging into liquid ethane or liquid nitrogen. Grids are then loaded into a TEM operating at cryogenic temperatures. Crystals are located on the grid and screened for initial diffraction quality. Continuous rotation MicroED data are collected from a subset of the screened crystals, where the data are saved using a fast camera as a movie

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1. Preparing small molecule samples

  1. Transfer a small amount (0.01 - 1 mg) of powder, liquid, or solids into a small vial or tube.
  2. For samples already in powder form, seal the tube using the cap until the sample is needed. Dry the liquid samples into powders prior to attempts at method 1 (step 3) or 2 (step 4).
    ​NOTE: Samples dissolved in liquid may use method 3 (5.X) below

2. Preparing TEM grids

NOTE: Some TEMs with autoloader.......

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MicroED is a cryoEM method that leverages the strong interactions between electrons and matter, which allows for the investigation of vanishingly small crystals12,13. After these steps, it is expected to have a diffraction movie in crystallographic format collected from microcrystals (Movie 1). Here, the technique is demonstrated using carbamazepine12. The results show a continuous rotation MicroED dataset from a carbamaze.......

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Sample preparation is typically an iterative process, where optimizations are made after sessions of screening and data collection. For small-molecule samples, it is often prudent to first attempt grid preparation without glow-discharging the grids, since many pharmaceuticals tend to be hydrophobic10,11. If the grids have too few nanocrystalline deposits, it is a good idea to try again after first glow-discharging the grids. It may be the case that the crystals f.......

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The Gonen lab is supported by funds from the Howard Hughes Medical Institute. This study was supported by the National Institutes of Health P41GM136508.


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Name Company Catalog Number Comments
0.1-1.5mL Eppendorf tubes Fisher Scientific 14-282-300 Any vial or tube will do.
Autogrid clips Thermo-Fisher 1036173 Clipped grids are not required for MicroED. They are required for Thermo-Fisher TEMs equipped with an autoloader system.
Autogrid C-rings Thermo-Fisher 1036171
Carbamazapine Sigma C4024-1G Any amount will suffice for these experiments
CMOS based detector Thermo-Fisher CetaD 16M We used a CetaD 16M, but any detector with rolling shutter mode or sufficiently fast readout is acceptable. 
Delphi software Thermo-Fisher N/A Software on Thermo-Fisher TEM systems that allows for manual rotation of the sample stage
EPU-D software Thermo-Fisher N/A Commercial software for the acquisition of MicroED data
Glass cover slides Hampton HR3-231
Glow discharger Pelco easiGlow
High PrecisionTweezers EMS 78325-AC Any high precision tweezer will do
Liquid nitrogen vessel Spear Lab FD-800 A standard foam vessel for handling specimens under liquid nitrogen - 800mL
SerialEM software UC Boulder N/A Free software distributed by D. Mastronarde. Department of Molecular, Cellular, and Developmental Biology
TEM grids Quantifoil/EMS Q310CMA Multi-A 300 mesh grids were used here, but any thin carbon grids will work. For these small molecules, we suggest starting with continuous carbon. 
transmission electron microscope (TEM) Thermo-Fisher Talos Arctica
Whatman circular filter paper Millipore-Sigma WHA1001090 90mm or larger

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