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Near-Atomic Resolution Cryo-EM Image Reconstruction of RNA

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RNA Structure and Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2568))

Abstract

The rapid development of cryogenic electron microscopy (cryo-EM) enables the structure determination of macromolecules without the need for crystallization. Protein, protein–lipid, and protein–nucleic acid complexes can now be routinely resolved by cryo-EM single-particle analysis (SPA) to near-atomic or atomic resolution. Here we describe the structure determination of pure RNAs by SPA, from cryo-specimen preparation to data collection and 3D reconstruction. This protocol is useful to yield many cryo-EM structures of RNA, here exemplified by the Tetrahymena L-21 ScaI ribozyme at 3.1-Å resolution.

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References

  1. Zhang K, Pintilie GD, Li S et al (2020) Resolving individual atoms of protein complex by cryo-electron microscopy. Cell Res 30:1136–1139

    Article  Google Scholar 

  2. Zhang K, Li S, Pintilie G, et al (2020) A 3.4-Å cryo-electron microscopy structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles. QRB Discov 1:e11

    Google Scholar 

  3. Roh S-H, Shekhar M, Pintilie G et al (2020) Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of V complex. Sci Adv 6

    Google Scholar 

  4. Cogan DP, Zhang K, Li X et al (2021) Mapping the catalytic conformations of an assembly-line polyketide synthase module. Science 374:729–734

    Article  CAS  Google Scholar 

  5. Zheng SQ, Palovcak E, Armache J-P et al (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331–332

    Article  CAS  Google Scholar 

  6. Li X, Mooney P, Zheng S et al (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590

    Article  CAS  Google Scholar 

  7. Cheng A, Tan YZ, Dandey VP et al (2016) Strategies for automated CryoEM data collection using direct detectors. Methods Enzymol 579:87–102

    Article  CAS  Google Scholar 

  8. Punjani A, Rubinstein JL, Fleet DJ et al (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296

    Article  CAS  Google Scholar 

  9. Chen M, Bell JM, Shi X et al (2019) A complete data processing workflow for cryo-ET and subtomogram averaging. Nat Methods 16:1161–1168

    Article  CAS  Google Scholar 

  10. Scheres SHW (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530

    Article  CAS  Google Scholar 

  11. Scheres SHW (2016) Processing of structurally heterogeneous Cryo-EM data in RELION. Methods Enzymol 579:125–157

    Article  CAS  Google Scholar 

  12. Zhao Y, Schmid MF, Frydman J et al (2021) CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin. Nat Commun 12:4754

    Article  CAS  Google Scholar 

  13. Zhang K, Zhang H, Li S et al (2019) Cryo-EM structures of vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution. Proc Natl Acad Sci USA 116:6800–6805

    Article  CAS  Google Scholar 

  14. Punjani A, Fleet DJ (2021) 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J Struct Biol 213:107702

    Article  CAS  Google Scholar 

  15. Zhang K, Zheludev IN, Hagey RJ et al (2021) Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nat Struct Mol Biol 28:747–754

    Article  CAS  Google Scholar 

  16. Kappel K, Zhang K, Su Z et al (2020) Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures. Nat Methods 17:699–707

    Article  CAS  Google Scholar 

  17. Zhang K, Li S, Kappel K et al (2019) Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution. Nat Commun 10:5511

    Article  CAS  Google Scholar 

  18. Su Z, Zhang K, Kappel K et al (2021) Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution. Nature 596:603–607

    Article  CAS  Google Scholar 

  19. Tian S, Das R (2017) Primerize-2D: automated primer design for RNA multidimensional chemical mapping. Bioinformatics 33:1405–1406

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Tian S, Yesselman JD, Cordero P et al (2015) Primerize: automated primer assembly for transcribing non-coding RNA domains. Nucleic Acids Res 43:522–526

    Article  Google Scholar 

  21. Rohou A, Grigorieff N (2015) CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216–221

    Article  Google Scholar 

  22. Tang G, Peng L, Baldwin PR et al (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157:38–46

    Article  CAS  Google Scholar 

  23. Emsley P, Lohkamp B, Scott WG et al (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501

    Article  CAS  Google Scholar 

  24. Adams PD, Afonine PV, Bunkóczi G et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221

    Article  CAS  Google Scholar 

  25. Pintilie G, Zhang K, Su Z et al (2020) Measurement of atom resolvability in cryo-EM maps with Q-scores. Nat Methods 17:328–334

    Article  CAS  Google Scholar 

  26. Lawson CL, Kryshtafovych A, Adams PD et al (2021) Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge. Nat Methods 18:156–164

    Article  CAS  Google Scholar 

  27. Torabi S-F, Chen Y-L, Zhang K et al (2021) Structural analyses of an RNA stability element interacting with poly(A). Proc Natl Acad Sci USA 118

    Google Scholar 

  28. Zhang K, Keane SC, Su Z et al (2018) Structure of the 30 kDa HIV-1 RNA dimerization signal by a hybrid Cryo-EM, NMR, and molecular dynamics approach. Structure 26:490–498. e3

    Article  CAS  Google Scholar 

  29. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

    Article  Google Scholar 

  30. Henderson R (2013) Avoiding the pitfalls of single particle cryo-electron microscopy: Einstein from noise

    Google Scholar 

Download references

Acknowledgment

This work is support by National Institutes of Health grants (P41GM103832 and R21AI145647 to WC) and start-up funding by the University of Science and Technology of China (KY9100000032 and KJ2070000080 to KZ). We thank Dr. Michael F. Schmid for helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. MOE Key Laboratory for Cellular Dynamics and Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Shanshan Li & Kaiming Zhang

  2. Department of Bioengineering, James H. Clark Center, Stanford University, Stanford, CA, USA

    Shanshan Li, Kaiming Zhang & Wah Chiu

  3. CryoEM and Bioimaging Division, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA

    Wah Chiu

Authors
  1. Shanshan Li
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  2. Kaiming Zhang
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  3. Wah Chiu
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Corresponding author

Correspondence to Wah Chiu .

Editor information

Editors and Affiliations

  1. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Jienyu Ding

  2. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Jason R. Stagno

  3. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Yun-Xing Wang

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© 2023 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply

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Li, S., Zhang, K., Chiu, W. (2023). Near-Atomic Resolution Cryo-EM Image Reconstruction of RNA. In: Ding, J., Stagno, J.R., Wang, YX. (eds) RNA Structure and Dynamics. Methods in Molecular Biology, vol 2568. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2687-0_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2687-0_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2686-3

  • Online ISBN: 978-1-0716-2687-0

  • eBook Packages: Springer Protocols

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