![]() To date, there are only three protein-free RNA cryo-EM structures determined at 4 Å or better resolution (Table 1), which accounts for 0.02% of the entire 17349 depositions in EMDB (Fig. 24, 25 Although cryo-EM SPA has extended our accessibility to more challenging biological structures and systems, protein-free RNA structures accrue at a much slower rate compared to proteins and protein-nucleic acid complexes in the Electron Microscopy Data Bank (EMDB). The lack of RNA structures is most likely due to the intrinsic heterogeneity of RNAs caused by flexible ribose and phosphate backbone, weak long-range tertiary interactions, alternative conformations, and dynamics among multiple functional states, which pose great challenges to X-ray crystallography to obtain RNA crystals with high-resolution diffraction information, and to NMR to solve structures of RNAs larger than 100 nucleotides (100 nt). There are currently 1569 protein-free RNA and 9790 protein-nucleic acid complex structures deposited in the PDB that accounts for only about 6% of the entire PDB deposition (Fig. 23 In contrast to our apparently better understanding of protein structures and functions, our knowledge of RNA structures remains scarce. 21, 22 In the Protein Data Bank (wwPDB), there are currently 183980 depositions of protein complexes, which has facilitated the development of accurate protein structure prediction algorithms. 17, 18, 19 It is estimated that about 85% of the human genome is transcribed into RNA, 20 with more than 80% of the genome estimated to be biologically and functionally relevant, 21 however, only 1.5 percent of the genome encodes proteins. RNA plays an essential role in various important biological processes by folding and sustaining a three-dimensional (3D) structure in order to perform functions such as catalysis and gene regulation. 13 Readers interested in technical developments and current challenges in SPA are directed to a number of comprehensive reviews for further details. Whereas the above features make SPA particularly useful to determine structures of larger proteins and protein complexes (>200 kDa), additional technical advances, especially the development of Volta phase plate (VPP), 12 have pushed the limit of protein molecular weight to as low as 52 kDa for structure determination by SPA. 9 Because cryo-EM SPA is featured by minimal amount of specimen under near-native crystal-free condition, automated data collection with continually increasing throughput, 10 and comprehensive data processing pipelines capable of resolving structural heterogeneity, 11 it has become a widely adopted structural biology technique for structural biologists whose portfolio used to have only X-ray crystallography and NMR. 3, 4, 5 Aside from single-particle analysis (SPA) that has gained tremendous popularity, other cryo-EM approaches have also benefited from the revolution, such as cryo-electron tomography (cryo-ET), 6, 7 micro-electron diffraction (MicroED) 8, and cryo-scanning transmission electron microscopy (cryo-STEM). 1, 2 More recently, several studies have achieved atomic-resolution cryo-EM reconstruction using either the next-generation hardware or the most up-to-date commercially available setup. The recent resolution revolution triggered by the development of direct electron detectors and other technical advances has allowed cryo-EM to break the previous resolution barrier(s) and led to exponential growth of near-atomic cryo-EM structures.
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