Genomics and Applied Biology 2024, Vol.15, No.1, 27-38 http://bioscipublisher.com/index.php/gab 30 2 The Advantages of Cryo Electron Microscopy Technology in Protein Structure Analysis 2.1 High resolution imaging capability The advantage of cryo electron microscopy technology in protein structure analysis is first reflected in its high-resolution imaging ability. Although traditional X-ray crystallography methods can also provide information on protein structure, their resolution is often limited by factors such as crystal quality and radiation damage. And cryo electron microscopy technology can image individual protein molecules or complexes under conditions close to physiological conditions, without the need for crystallization, thus avoiding the limitations of crystal formation (Figure 2). Figure 2 Technical differences between X-ray crystallography and single particle cryo electron microscopy (Wang and Wang, 2017) Note: A: X-ray crystallography solves the physical and mathematical principles of structure; B: The physical and mathematical principles of EM in solving structures Freezing electron microscopy utilizes low-temperature rapid freezing technology to fix protein samples in structures close to their physiological states, effectively reducing structural changes during sample preparation. Mielanczyk et al. (2014) focused on different freezing preparation methods, starting with sample size dependent vitrification methods, followed by cryo FIB as a potential alternative to cryo electron microscopy (CEMOVIS), as well as discussions on cryo substitution and resin embedding for structural analysis. This demonstrates the crucial role of cryo EM and low-temperature rapid freezing technology in sample preparation, providing electron microscopy with near native sample observations. By combining advanced electron microscopy technology and image processing algorithms, cryo electron microscopy can achieve atomic level resolution and clearly reveal the fine structure of protein molecules. Yip et al. (2020) reported the use of a newly developed electron microscope to obtain a 1.25 Å resolution apoferrin structure. This result is almost twice the 3D information content of the current world record reconstruction (1.54 Å resolution). This study demonstrates significant progress made by cryo electron microscopy in structural biology, which now allows for direct visualization of individual atoms in proteins. The high-resolution imaging capability enables cryo electron microscopy to capture subtle structural features within protein molecules, such as the orientation of amino acid side chains, arrangement of hydrogen bonds, and intermolecular interactions. Liu and Yeates (2019) designed protein skeletons to bind and symmetrically display 12 small 26 kDa proteins (green fluorescent protein GFP), achieving cryo EM imaging at 3.8 Å resolution. This modular protein skeleton design can bind and display multiple proteins through small amino acid sequence changes. The high-resolution imaging ability of cryo electron microscopy technology also enables its application in a wider range of protein system studies. Whether it is prokaryotes or eukaryotes, whether it is a single protein or a large complex, cryoelectron microscopy can provide high-resolution structural information. This provides a powerful
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