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P-Type ATPases pp 443-455 | Cite as

Two-Dimensional Crystallization of Gastric H+,K+-ATPase for Structural Analysis by Electron Crystallography

  • Kazuhiro AbeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1377)

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.

Key words

Gastric proton pump H+,K+-ATPase Electron crystallography Cryo-electron microscopy Structural analysis Membrane protein Two-dimensional crystal 

Notes

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Cellular and Structural Physiology InstituteNagoya UniversityNagoyaJapan
  2. 2.Department of Medicinal ScienceGraduate School of Pharmaceutical Sciences, Nagoya UniversityNagoyaJapan

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