title: Two-Dimensional Crystallization of Gastric H(+),K(+)-ATPase for Structural Analysis by Electron Crystallography.
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.
authors.affiliation: Cellular and Structural Physiology Institute, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.|Department of Medicinal Science, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Abe
mesh_terms: D000818:Animals; D003460:Crystallization / Q000379:methods*; D003956:Dialysis; D008854:Microscopy, Electron; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D012995:Solubility; D013552:Swine
chemical_list: D000254:Sodium-Potassium-Exchanging ATPase
keywords: Cryo-electron microscopy; Electron crystallography; Gastric proton pump; H+,K+-ATPase; Membrane protein; Structural analysis; Two-dimensional crystal