Abstract
Electron crystallography of two-dimensional (2D) crystals has provided important information on the structural biology of P-type ATPases. Here, I describe the procedure for making 2D crystals of gastric H+,K+-ATPase purified from pig stomach. The 2D crystals are produced by dialyzing detergent-solubilized H+,K+-ATPase mixed with synthetic phospholipids. Removal of the detergent induces the reconstitution of H+,K+-ATPase molecules into the lipid bilayer. In the presence of fluorinated phosphate analogs, or in combination with transporting cations or the specific antagonist SCH28080, H+,K+-ATPase forms crystalline 2D arrays. The molecular conformation and morphology of the 2D crystals vary depending on the crystallizing conditions. Using these 2D crystals, three-dimensional structures of H+,K+-ATPase can be generated by data correction from ice-embedded 2D crystals using cryo-electron microscopy, followed by processing the recorded images using electron crystallography methods.
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Acknowledgements
The author acknowledges Drs. Tomohiro Nishizawa, Kazutoshi Tani, and Yoshinori Fujiyoshi for their contributions to the development of the 2D H+,K+-ATPase crystallization procedure. This work was supported by Grants-in-Aid for Young Scientist (A) and Platform for Drug Design, Discovery, and Development from METI, Japan.
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Abe, K. (2016). Two-Dimensional Crystallization of Gastric H+,K+-ATPase for Structural Analysis by Electron Crystallography. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_39
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_39
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
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