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Purification of Single-Stranded RNA Bacteriophages and Host Receptors for Structural Determination Using Cryo-Electron Microscopy

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Phage Engineering and Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2793))

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Abstract

Single-stranded RNA bacteriophages (ssRNA phages) are small viruses with a compact genome (~3–4 kb) that infect gram-negative bacteria via retractile pili. These phages have been applied in various fields since their discovery approximately 60 years ago. To understand their biology, it is crucial to analyze the structure of mature virions. Cryo-electron microscopy (cryo-EM) has been employed to determine the structures of two ssRNA phages, MS2 and Qβ. This chapter presents a method for purifying these two phages and their receptor, the F-pilus, to allow examination using cryo-EM.

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Acknowledgements

We would like to express our gratitude to Prof. Ry Young at the Texas A&M University, TX, for graciously granting us the pMS2000. We extend our sincere thanks to Prof. Peter J. Christie at McGovern Medical School, Houston, TX, for generously providing us with pOX38 as a kind gift. Additionally, we would like to acknowledge Zachary Lill, a lab member, for his invaluable assistance in proofreading. We are grateful for the support received from TAMU T3 and X-grants, the Welch Foundation grant A-1863, NSF grant MCB-1902392, and NIH grants R21AI156846 and R01GM141659, as well as funding from the Center for Phage Technology, jointly sponsored by Texas AgriLife and Texas A&M University.

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Authors and Affiliations

  1. Center for Phage Technology, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA

    Jirapat Thongchol & Junjie Zhang

Authors
  1. Jirapat Thongchol
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  2. Junjie Zhang
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Correspondence to Junjie Zhang .

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Editors and Affiliations

  1. Cellular Signaling Laboratory International Research, Center for Sensory Biology and Technology of MOST, Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China

    Huan Peng

  2. College of Life Science and Technology, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China

    Jianfeng Liu

  3. Department of Chemical and Biomolecular Engineering, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Irene A. Chen

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Thongchol, J., Zhang, J. (2024). Purification of Single-Stranded RNA Bacteriophages and Host Receptors for Structural Determination Using Cryo-Electron Microscopy. In: Peng, H., Liu, J., Chen, I.A. (eds) Phage Engineering and Analysis. Methods in Molecular Biology, vol 2793. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3798-2_13

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  • DOI: https://doi.org/10.1007/978-1-0716-3798-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3797-5

  • Online ISBN: 978-1-0716-3798-2

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