Structural Biology Technology Review|How MicroED, Cryo-electron Microscopy SPA, and X-ray Crystallography Complement Each Other

Structural biology is a discipline based on molecular biology, biochemistry, and biophysics, and the research objects are biological macromolecules, such as proteins and nucleic acids. The purpose is to use biological or physical approaches to understand the fine morphology of biological macromolecules, to explain the mechanisms of biological macromolecules’ driving functions, and to help biologists better understand the process of life activities. Moreover, most of the targets of drug molecules are proteins, therefore structural biology plays an important guiding role in drug research and development, bringing a clear understanding of mechanism study to drug development.

For the study of proteins in biological macromolecules, the main research tools are nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy (cryo-EM). NMR is mainly aimed at samples with small molecular weights (about 20 kDa) in solution and has not been used much in recent years. In recent years, new techniques have also emerged. Microcrystalline electron diffraction (MicroED) is a technology that uses cryo-electron microscopy to resolve the structure of nanocrystals, which can resolve nanocrystalline structures that are difficult to handle by X-ray crystallography because the electron beam interacts with the matter much more strongly than X-rays. In particular, for protein samples that were previously difficult to culture as single crystals, MicroED offers structural biologists a promising new tool to bridge the gap between existing techniques. The technology was named one of the top 10 breakthrough technologies of 2018 by the journal Science. 

| X-Ray Diffraction - The most traditional way of structure analysis

The discovery of X-rays by W. C. Roentgen as early as 1895 has driven the development of modern biology and has had a profound impact on the entire field of science and technology. In 1912, M. V. Laue, together with W. Friedrich and P. Knipping, a doctoral student of Röntgen, performed diffraction experiments on copper sulfate crystals using X-rays and discovered X-ray crystal diffraction by obtaining some coarse, elliptical spots on the negatives. Later, they performed X-ray diffraction experiments on ZnS, PbS, and NaCl crystals and obtained clear quadruple symmetric diffraction patterns. Laue also proposed the Laue equation to describe the X-ray diffraction of crystals. Soon after the publication of the X-ray diffraction photographs of ZnS crystals, W. L. Bragg repeated the experiment and derived the X-ray diffraction equation, the famous Bragg equation, in 1913. The discovery of X-ray diffraction and the establishment of the Lauer and Bragg equations marked the birth of X-ray crystallography. By 1957, X-ray crystallography had resolved the structure of the first biological macromolecule, sperm whale myoglobin. And up to now, about 150,000 structures of biological macromolecules have been resolved using X-ray crystallography. The reason why X-ray crystallography has developed so rapidly to resolve the 3D structure of proteins is mainly due to the following advantages: 1. it can achieve high resolution, close to the atomic level; 2. there is no restriction on the size and composition type of proteins, so both multi-subunit complexes with very large molecular weights and peptides with very small molecular weights can obtain ideal results by this method. However, the second advantage of this method is also its limitation, as it is often difficult to obtain good quality crystals for objects with large molecular weights or flexible structures.

|  Cryo-electron microscopy-based single particle analysis technique

In recent decades, cryo-electron microscopy has developed rapidly and gradually played a pivotal role in the field of structural biology. As early as 1939, Siemens manufactured the world's first commercially available electron microscope. To prevent damage to the sample by high vacuum and intense electron bombardment, the sample was treated with heavy metal staining or sugar embedding. However, the resolution is limited by the destructive treatment of the samples and the observation at room temperature. By 1984, J. Dubochet et al. developed a method for freezing biological samples and summarized the method for obtaining glassy ice (1,2). This was the first rapid freezing technique (cryo-freezing) and ushered in the era of "cryo" electron microscopy. By rapidly plunging a solution sample of biomolecules into liquid ethane cooled to the temperature of liquid nitrogen, a frozen sample with a suitable ice thickness was obtained, and then the uniformly distributed biomolecule particles in the ice were photographed, and thousands of photographs were obtained. The two-dimensional photographs are analyzed by an algorithm to obtain the final structure, which is called single-particle analysis. In recent years, with the advent of direct detection devices (DDD) and the development of various data processing software, the proportion of three-dimensional structures of biological macromolecules resolved by cryo-electron microscopy is increasing (see Figure 1). For example, a study published in Science in March 2020 determined the 3.7 Å and 3.9 Å structures of human-derived glucagon receptor (GCGR) bound to glucagon and different classes of heterotrimeric G proteins (Gs or Gi1) by cryo-electron microscopy single-particle analysis techniques (see Figure 2). The multiple structures from this study combined with pharmacological data provide important insights for the treatment of type II diabetes and obesity (3).

Figure 1. Trends in the number of three-dimensional structures of biological macromolecules resolved by cryo-electron microscopy.
Figure 2. Structures of Glucagon-GCGR-GS-Nb35 & glucagon-GCGR-Gi1-Scfv16 resolved by cryo-electron microscopy.

The single-particle technique does not require crystals to be obtained and uses very small amounts of samples, which makes it possible to study membrane proteins or macromolecular complexes that are difficult to crystallize. However, while single-particle techniques require a lower volume of samples, they do not reduce the high demand for sample homogeneity. Moreover, although with the continuous improvement of detectors and algorithms, single-particle analysis techniques have been able to obtain structures with a resolution of up to 2 Å4, it is still relatively an isolated case, and its resolution is still generally around 3 Å. However, the reason why human beings can create civilization lies in the never-ending search for mysterious nature and scientific truth; and the continuous development and advancement of various research fields will inevitably influence and promote each other.

|  MicroED: Crystallography and cryo-electron microscopy in a contemporary blend

At the beginning of the development of electron microscopy, X-ray crystallography techniques were flourishing; this was also the time when diffraction theory and data processing methods were maturing. Electron crystallography is the product of combining electron microscopy techniques with crystallography. In 2007, scientists at Stockholm University cleverly combined crystallography and electron microscopy by using cryo-electron microscopy to look at crystals and developed an important new technique, MicroED (Micro electron diffraction). MicroED is a method of diffracting tiny crystals by electrons, and then collecting electron diffraction data and analyzing protein structures. Because of the short wavelength of electrons, they are strongly scattered by matter (atoms scatter electrons 10,000 times more strongly than X-rays), so they can achieve a higher resolution. Moreover, it requires very small crystal sizes, with submicron-sized crystals producing sufficiently high signal-to-noise diffraction signals. These unique advantages are certainly good news for those samples where tiny crystals can be obtained and the size cannot be optimized to become larger. This technique was also named one of the top 10 technological breakthroughs of 2018 by the journal Science 2018. In the early days of MicroED development, model samples such as lysozyme, catalase, and Ca2+-ATPase were resolved to atomic resolution. Later, MicroED further developed and was no longer limited to the model samples, but also resolved structures of unknown proteins such as α-synuclein central peptide and R2loxase6 (see Figure 3), all with resolutions up to ~1 Å. After 2016, MicroED developed rapidly and the number of resolved protein structures increased rapidly (see Figure 4), with prions and FUS LC (fused in sarcoma low-complexity domain) and other samples have obtained crystal structures with a high resolution of ~1 Å (see Figure 4). And then MicroED resolved progressively more difficult objects, even including membrane proteins such as complexes and ion channels (see Figure 4). Starting from 2018, it has been possible to combine MicroED with cryo-Focused Ion Beam (cryo-FIB) technology, which can thin crystals of slightly larger size but still not applicable with X-ray crystallography, and expand the range of crystal sizes applicable to MicroED to tens of microns, greatly widening the MicroED technology The combination of cryo-FIB and X-ray crystallography allows for the thinning of crystals that are slightly larger but still not suitable for X-ray crystallography.

Figure 3. MicroED resolved the R2lox structure.

Figure 4. Study statistics of MicroED.

|  Simple process, high resolution, short time - MicroED opens up new ways of exploring structural biology

The principle of MicroED is similar to that of X-ray crystallography. Like X-rays, electron waves can be diffracted when irradiated on a crystal-like sample. When the objective plane of the intermediate mirror is located at the back focus of the objective, the diffraction spectrum of electrons magnified by the intermediate mirror and the projection mirror will be obtained on the fluorescent screen, which is the diffraction mode of transmission electron microscopy. In the actual transmission electron microscope, the current of the intermediate mirror for imaging mode and diffraction mode has been set, and the two modes can be switched by the corresponding button (Diffraction button). MicroED is a good complement to the field of structural biology.

Due to the unique advantages of MicroED, in addition to biological macromolecules, it is more than suitable for the study of chemical small molecules, natural products, and pharmaceutical preparations. Because small chemical molecules are in powder form, their size fits well with the scope of MicroED research, and structural information can be obtained from the powder basically within a few hours. Many successful cases of using MicroED to study chemical small molecules have demonstrated the efficiency and applicability of this technique (see Figure 6). MicroED research on biological macromolecules and small molecule drugs has demonstrated its great potential for structural biology, providing a promising new tool for structural biologists.

Figure 5. Workflow of MicroED(7).

Figure 6. Case of chemical small molecules and natural products obtained by MicroED (7).

In general, both X-ray crystallography and cryo-electron microscopy single-particle techniques, as well as the emerging MicroED technology, are gradually advanced by scientists in continuous experimentation and optimization. They complement each other in terms of technology and research scope and promote each other as much as possible to bring humans infinitely close to the natural truth and continue to lead the development of life sciences. For commercial structural biology testing services, ReadCrystal has the international advanced testing platform of MicroED and also provides SCXRD and CryoEM-SPA testing services.

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2. Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W. Cryo-electron microscopy of viruses. Nature 308, 32-36 (1984).
3. Qiao, A. & Han, S. Structural basis of G(s) and G(i) recognition by the human glucagon receptor.   367, 1346-1352 (2020).
4. Yip, K. M. F., N. Atomic-resolution protein structure determination by cryo-EM.   587, 157-161 (2020).
5. Shi, D., Nannenga, B. L., Iadanza, M. G. & Gonen, T. Three-dimensional electron crystallography of protein microcrystals. Elife 2, e01345 (2013).
6. Xu, H. et al. Solving a new R2lox protein structure by microcrystal electron diffraction. Science Advances 5 (2019).
7. Nguyen, C. & Gonen, T. Beyond protein structure determination with MicroED. Current Opinion in Structural Biology 64, 51-58 (2020).