Chapter title |
Two-Dimensional Crystallization of Gastric H + ,K + -ATPase for Structural Analysis by Electron Crystallography
|
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Chapter number | 39 |
Book title |
P-Type ATPases
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Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3179-8_39 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3178-1, 978-1-4939-3179-8
|
Authors |
Kazuhiro Abe |
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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