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 movie3. These movies are converted to a standard crystallographic format and processed almost identically as an X-ray crystallography experiment4.
MicroED was originally developed to investigate protein microcrystals1,2. A bottleneck in protein crystallography is growing large, well-ordered crystals for traditional synchrotron X-ray diffraction experiments. As electrons interact with matter orders of magnitude stronger than X-rays, the limitations of the crystal size needed to produce detectable diffraction is considerably smaller5. Additionally, the ratio of elastic to inelastic scattering events is more favorable for electrons, suggesting that more useful data can be collected with a smaller overall exposure5. Constant developments have allowed for MicroED data to be collected from the most challenging microcrystals6,7,8,9.
Recently, MicroED has been shown to be a powerful tool for determining the structures of small molecule pharmaceuticals from apparently amorphous materials10,11,12,13. These powders can come straight from a bottle of purchased reagent, a purification column, or even from crushing a pill into a fine powder10. These powders appear amorphous by eye, but may be either entirely composed of nanocrystals or merely contain trace amounts of nanocrystalline deposits in a greater non-crystalline, amorphous fraction. Application of the material to the grid is facile, and the subsequent steps of crystal identification, screening, and data collection might even be automated in the near future14. While others may use different methods for sample preparation and data collection, here the protocols developed and used in the Gonen laboratory for preparing samples of small molecules for MicroED and for data collection are detailed.
1. Preparing small molecule samples
2. Preparing TEM grids
NOTE: Some TEMs with autoloader systems require that the grids be clipped and placed into a cassette prior to loading into the TEM column. Clipping involves physically securing the 3 mm TEM grid into a metal ring that the autoloader can manipulate. This step and subsequent steps can be performed using either normal TEM grids, or TEM grids that have been clipped. For these experiments, it is often easier to manipulate the grids if they have been clipped ahead of time.
3. Applying sample to grids by creating a homogenous fine powder (Method 1)
4. Applying sample to grids by applying the "shaking" method (Method 2)
5. Applying sample to grids using the evaporation method (Method 3)
6. Freezing and loading grids into the TEM
7. Collecting MicroED data
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 carbamazepine microcrystal identified on a TEM grid (Movie 1). A good dataset has strong, clear spots that are not smeared or split, and has only a single lattice on each frame that can easily be followed by stepping through the movie19. These data are easily indexed, integrated, and scaled using standard X-ray crystallography software4. Split spots can be seen from a crystal that has been cracked, and two orientations of the same crystal are closely aligned, but not quite coincident19. Multiple lattices can also occur, particularly for these small-molecule crystals, where multiple single crystals have stuck together in a clump on the grid. Another common scenario occurs where the crystals have been frozen incorrectly or treated too harshly during fragmentation, and no diffraction is observed9.
After data collection, integration, and structure solution, it is expected that a high-resolution structure is determined (Figure 5). Obtaining a clear structure solution will ultimately depend on the quality and completeness of the data.
Figure 1: Preparation of a pre-clipped TEM grid for small molecule investigation. (A) A tube with a small portion of sample for investigation. (B) Crushed sample between two microscope slides. (C) The carbon side of the pre-clipped grid, and (D) the copper side of the pre-clipped grid. (E) A pre-clipped TEM grid after the crushed powder has been dropped onto it. Scale bars 3mm in (C), (D), and (E). Please click here to view a larger version of this figure.
Figure 2: Identification of small molecule crystals in the TEM. (A) An all-grid atlas or montage at low magnification. (B) A single low magnification image used for screening. (C) Higher magnification image used to identify smaller grains. (D) High magnification micrograph of a clumped small molecule crystal. Scale bar 750 µm in (A), 50 µm in (B), 10 µm in (C), 1 µm in (D). Please click here to view a larger version of this figure.
Figure 3: Screening and aligning microcrystals for MicroED data collection. (A) High magnification micrograph of a microcrystal. (B) Micrograph of the isolated crystal within the selected area aperture. (C) The same microcrystal in the aperture with the stage tilted to -69 °. Scale bars all 1 µm. Please click here to view a larger version of this figure.
Figure 4: Examples of MicroED data. (A) high quality MicroED data with clear, sharp spots suitable for high-resolution structure determination. (B) Weak, smeared MicroED data with poor lattice definition. In this example the alignment is also off so the diffraction is only apparent on one side of the image (C) Poor MicroED data showing multiple lattices and split and/or smeared spots. Inset of blue area enhanced in bottom right showing smeared, split spots. Please click here to view a larger version of this figure.
Figure 5: MicroED structure of Carbamazepine. Atomic model shown as sticks with carbon atoms colored white, oxygen red, and nitrogen blue. The 2Fo-Fc map is contoured at the 1.5 σ level and colored blue. The Fo-Fc map showing hydrogens is contoured at the 3.0 σ level and colored green. This figure was adapted from the deposited maps of EMDB-928410. Please click here to view a larger version of this figure.
Movie 1: MicroED data set from carbamazepine. Dataset spans almost 90˚, from -68° to +20°. Each diffraction pattern spans a wedge of 0.5° in reciprocal space and corresponds to an exposure of 1s at an exposure rate of 0.01 e– Å-2 s-1. Please click here to download this Movie.
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 from lyophilized powders are too large and thick to collect good data. In these cases, it may be possible to collect data from an edge or thinner part of a larger crystal. If this proves difficult, grinding the powder down to a finer consistency using a rougher surface, such as a mortar and pestle may be necessary.
MicroED data are typically collected with the TEM operating in microprobe mode4,20. Here, the size of the TEM beam that corresponds to an exposure rate of 0.01 e– Å-2 s-1 is typically around 10 µm in diameter, which is much larger than the typical microcrystals1,21. The signal is then isolated from the crystals of interest using a selected area aperture (Figure 3)2,20. Various aperture sizes allow for quick adjustment of the setup to varying sizes of crystals. Alternatively, it is possible to collect data with the TEM operating in nano probe mode. This reduces the size of the beam by approximately a factor of 5. A smaller beam corresponds to a commensurately higher exposure rate in the beam footprint. Since many TEMs are two condenser lens systems, the parallel condition will dictate that the beam be a single size in either microprobe or nano probe mode. Reaching an exposure rate of 0.01 e– Å-2 s-1 in nano probe without adjusting the gun lens is challenging. The choice between the two is up to the user. An advantage of nano probe is that there is less of a need to insert and retract the selected area aperture between screening in imaging and diffraction modes of operation. However, with modern microscopes insertion and retraction of the SA aperture is automatic and accurate. Microprobe offers larger flexibility in isolating diffraction by having access to multiple sizes of selected area apertures. The larger beam in microprobe may also expose nearby crystals, whereas nano probe can more precisely target individual crystals.
The presented protocol is the standard approach to MicroED data collection for small molecules used in our laboratory10,11,12,13. There are many adaptations and modifications that could be implemented. The best approach to making grids with high crystal density is most dependent on the familiarity of the user with a given approach. There are many cases where drugs are present as large crystals that are too fragile to physically fragment without losing diffracting power19. In these cases, the recently adapted method of focused ion-beam milling to thin the crystals to make them more accessible to MicroED6,7,8,9,22.
The authors have nothing to disclose.
The Gonen lab is supported by funds from the Howard Hughes Medical Institute. This study was supported by the National Institutes of Health P41GM136508.
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 |