Genomics and Applied Biology 2024, Vol.15, No.1, 27-38 http://bioscipublisher.com/index.php/gab 33 dynamic changes. The application of cryo electron microscopy technology enables scientists to reveal the assembly methods, functional mechanisms, and interactions between subunits of complexes by providing high-resolution structural details, greatly deepening their understanding of these biological processes. Freezing electron microscopy technology is particularly suitable for capturing the structural changes of complex protein complexes during their biological function execution. These dynamic structural information provide valuable perspectives for understanding how complexes transition between different functional states. In addition, this technology can handle the heterogeneity of samples and identify different conformational states from a large number of images through single particle analysis technology, which is of great significance for studying the regulatory mechanisms and functional diversity of complexes. The unique ability of cryo electron microscopy to analyze asymmetric or extremely large protein complexes provides an unprecedented perspective to explore the secrets of these complex biological entities. Costa et al. (2017) described the method of using single particle cryo EM for studying the structure of biological complexes, which has now become a mature technology in structural biology competing with X-ray crystallography. The latest progress in EM enables us to determine the structure of protein complexes at a resolution of 3-5 Å, with a wide range of sizes ranging from approximately 200 kDa to hundreds of megadaltons. Schmidt and Urlaub (2017) explored a method for elucidating the structure of large protein complexes by combining cryo EM and cross-linking mass spectrometry (CX-MS). The proximity information between amino acid residues provided by CX-MS serves as a distance constraint for homology or de novo modeling, providing valuable information for addressing unclear areas in cryo EM images. Danev et al. (2019) emphasized the rise of cryo EM as a powerful structural determinant technology. Single particle analysis (SPA) is its most prolific branch used to determine high-resolution protein structures in laboratories worldwide. 3.3 Analysis of membrane proteins and dynamic protein structures Membrane proteins are the core components of biological cell functions, playing crucial roles in key processes such as signal transduction, substance transport, and intercellular interactions. However, due to the poor stability and low solubility of membrane proteins in lipid bilayers, traditional structural biology methods such as X-ray crystallography are difficult to obtain their high-resolution structures. Freezing electron microscopy technology has broken through this limitation and can directly observe the structure of membrane proteins in close proximity to their original ecological environment without the need for crystallization, providing extremely valuable information for drug design targeting these key biomolecules. Similarly, in the study of dynamic protein structure, cryo electron microscopy has also demonstrated its unparalleled ability. The function of these proteins usually depends on their ability to transition between different conformations, and cryoelectron microscopy can capture the instantaneous and transitional structures of these proteins during their functional execution. This ability to analyze dynamic protein structures provides a deeper perspective on how proteins regulate their function through structural changes, and opens up new avenues for developing new therapeutic methods, especially drugs that can precisely regulate these dynamic processes. Bonomi and Vendruscolo (2017) discussed how to use cryo EM to simultaneously determine the structure and dynamics of proteins, and provided an effective way to provide all this information in the form of structural sets through an integrated approach combining experimental and computational methods Nygaard et al. (2020) discussed the challenges and techniques of using single particle cryo EM structures to analyze small-sized membrane proteins, such as increasing size through the use of antibody fragments and providing markers for particle alignment, as well as some unresolved issues such as complex displacement at the air water interface. Thonghin et al. (2018) discussed the use of cryo EM for membrane protein structure in breakthrough studies, which can handle relatively small samples and tolerate the presence of detergents. These studies demonstrate the potential of cryo EM technology in revealing membrane proteins and dynamic protein structures, providing
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