CryoEM MicroED Methods to Determine Crystalline Structure

By Cvetelin Vasilev, Ph.D.Apr 24 2020

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Fast and accurate structural characterization is an important step in many scientific experiments and industrial manufacturing processes. Most of the properties of both chemical and biological materials depend critically on their molecular structure. Chemical structural elucidation is of particular importance when developing novel chemical compounds in several sectors such as biochemical, pharmaceutical and agrochemical industries. 

With the increasing complexity of the novel active substances, the successful structure characterization requires an array of complementary analytical techniques, which is often time-consuming.

A newly developed analytical method called microcrystal electron diffraction (MicroED), which is based on the well-established cryo-electron microscopy (CryoEM) technique, can rapidly determine the molecular structure of crystalline materials with nearly-atomic resolution.

Several well-established analytical methods accurately determine the molecular structure of different chemical compounds, such as mass spectrometry, ultraviolet-visible (UV-Vis) and infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. However, these methods are usually slow, indirect and sometimes require quite involved and lengthy sample preparation.

Several methods allow rapid identification of some specific organic molecules (such as drugs and explosives), but these cannot be employed when analyzing substances with previously unknown structures.

Problems with Existing Methods for Structural Analysis

In the past decades, the NMR spectroscopy, mass spectrometry and X-ray crystallography became the most predominant methods for structural characterization. These techniques are widely employed in both experimental chemistry and industrial research for structural elucidation of complex chemical molecules.

X-ray crystallography relies on the scattering of X-rays when interacting with the electron shells of the atoms. The resulting diffraction pattern is then interpreted and the arrangement and interconnectivity of the atoms and bond orientation within a given molecule can be resolved, providing fast and reliable structural characterization.

However, the method requires relatively large (at least a few hundred microns in size) and highly ordered crystals as a specimen (preparation of such crystals is a time-consuming task and might take several weeks). Besides, smaller atoms such as hydrogen with a small number of electrons in the shells, interact weakly with X-rays and because of that such atoms are not always detected reliably (their positions are often extrapolated).

The NMR spectroscopy and mass spectrometry are indirect methods, where the structure is determined by measuring the interaction of the atomic nuclei in the molecules with an external magnetic field and by determination of the mass of ionized molecular fragments, respectively. In both cases, the structure determination requires several lengthy steps of data processing and data matching to existing databases.

The complex and unreliable specimen preparation prevents the routine use of X-ray crystallography for industrial research (despite the fact the structural data provided is superior to any other method). This, combined with the low throughput and lack of structural accuracy of the NMR spectroscopy and mass spectrometry, motivate organic chemists from academia and industry to explore faster and more reliable methods for structural determination.

Researchers Take Advantage of Recent Breakthrough in CryoEM

Tamir Gonen and his team at the University of California in Los Angeles recently developed a method for structural analysis called MicroED based on the CryoEM technique, for the development of which the Nobel Prize in Chemistry was awarded in 2017. MicroED is capable of analyzing a very wide range of solid materials such as drugs and drug-like substances, vitamins, proteins and other organic and inorganic materials.

Tightly Focused Electron Beams Probe Nanocrystals

The most significant advantage of MicroED technique comes from the fact that the electron beam interacts 200 times stronger with the atoms compared to X-rays. This allows all the data to be collected much faster and on smaller crystals, as the electron beam can be easily focused and collimated to a diameter of less than one nanometre.

The specimens still need to be crystalline, but the size of the crystals can be very small, down to approximately 100 nanometres across (easily identified with an electron microscope), or a billion times smaller than the crystals analyzed by X-rays. This is a very important development, as it is much easier and quicker to obtain such small crystals that are defect-free. For example, this can be done by simple evaporation or recrystallization.

CryoEM Combined with Computed Tomography Pinpoint Atomic Positions

In the new method, the intensity of the electron beam can be decreased 200 times, which minimizes sample damage caused by the high-energy electron beam. The specimen crystals are scanned at different angles as the whole sample rotates at a speed of 0.5 degrees per second, allowing the use of existing computed tomography (CT) algorithms to reduce the error associated with multiple scattering of the electrons within the specimen.

These developments are underpinned by the groundbreaking advances in CryoEM instrumentation in the last 10 years, including:

• Automated sample manipulation
• Development of modern high-sensitivity direct electron detectors
• High-throughput automated data analysis software.

The above are key factors for the success of the novel method.

MicroED Rapidly Resolves Structures with Angstrom Resolution

Since the invention of the technique in 2013, numerous proteins, peptides and small-molecule structures have been resolved by using MicroED, including structures that resisted other methods.

As an excellent example of the capabilities of the new method, the mammalian steroid hormone progesterone was analyzed as received from a commercial supplier (a fine powder consisting of nanometre-sized crystals).

A small quantity of the seemingly amorphous solid was dispersed onto an electron microscope sample grid (a holey carbon copper grid), flash-frozen in liquid nitrogen and transferred to a cryo-electron microscope. The multi-angle diffraction data collected from a single nanocrystal (3 minutes overall acquisition time) was sufficient to determine the structure of the steroid with a resolution of 1Å. Even more remarkable is the fact that the entire process from the powder sample to the final refined structure took less than 30 minutes in total.

Fast and Accurate Structure Determination Proves MicroED’s Versatility

Encouraged by the performance of MicroED technique, the researchers went on to explore a wide range of substances (some with an already known structure) such as proteins, drug constituents and vitamins.

Large molecules (e.g. proteins) needed to be embedded in vitreous ice to minimize the thermal motion of the molecules that causes the diffraction patterns to blur, resulting in lower overall structure resolution. Small molecule crystals with densely packed molecules did not pose such problems. It is possible to obtain better-resolved structures when studying smaller molecules using the MicroED method.

Some of the obtained structures are summarized in Table 1. It is particularly striking to see the dramatic increase in the structural accuracy and resolution (down to a sub-angstrom level) resulting from the refinement and improvement of the method in the last 5-7 years, while at the same time allowing the investigation of substances with lower molecular weight.

New Prospects for Drug Discovery and Pharmaceutical Industry

Importantly, the MicroED technique scored highly when used to analyze over-the-counter medications, natural products (obtained from commercial sources and used as received) and even crudely purified substances from early-stage drug discovery research. It also provided very fast atomic resolution structure determination.

Another significant advantage of the new method is the ability to identify different constituents in a multi-component mixture with a significant structural heterogeneity and presence of impurities - a task in which both X-ray crystallography and NMR spectroscopy perform quite poorly.

Several powder substances, such as vitamins and alkaloids used in drugs were mixed on a sample grid, and MicroED data was collected from several of the mixed nanocrystals. Each species was identified within several minutes based on its diffraction pattern and unit cell parameters (each of the four substances was analyzed individually beforehand).

These astonishing capabilities bring the prospect of the wide use of the MicroED method across the research and industrial laboratories as a rapid and powerful tool for structural characterization and particularly well-suited for drug characterization and discovery.

References and Further Reading

Jones, C. G., et al. (2018) The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination. ACS Cent. Sci., 4, 1587-1592. Available at: https://doi.org/10.1021/acscentsci.8b00760

Nannenga, B. L. and Gonen, T. (2019) The cryo-EM method microcrystal electron

diffraction (MicroED). Nature Methods, 16, 369-379 Available at: https://doi.org/10.1038/s41592-019-0395-x

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