title: Carbon sandwich preparation preserves quality of two-dimensional crystals for cryo-electron microscopy.
abstract: Electron crystallography is an important method for determining the structure of membrane proteins. In this paper, we show the impact of a carbon sandwich preparation on the preservation of crystalline sample quality, using characteristic examples of two-dimensional (2D) crystals from gastric H(+),K(+)-ATPase and their analyzed images. Compared with the ordinary single carbon support film preparation, the carbon sandwich preparation dramatically enhanced the resolution of images from flat sheet 2D crystals. As water evaporation is restricted in the carbon-sandwiched specimen, the improvement could be due to the strong protective effect of the retained water against drastic changes in the environment surrounding the specimen, such as dehydration and increased salt concentrations. This protective effect by the carbon sandwich technique helped to maintain the inherent and therefore best crystal conditions for analysis. Together with its strong compensation effect for the image shift due to beam-induced specimen charging, the carbon sandwich technique is a powerful method for preserving crystals of membrane proteins with larger hydrophilic regions, such as H(+),K(+)-ATPase, and thus constitutes an efficient and high-quality method for collecting data for the structural analysis of these types of membrane proteins by electron crystallography.
authors.affiliation: Faculty of Science, Department of Biophysics, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-0852, Japan.
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Abe
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Tani
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujiyoshi
mesh_terms: D002244:Carbon / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D003461:Crystallography / Q000295:instrumentation / Q000379:methods; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000648:ultrastructure*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure*; D013048:Specimen Handling / Q000379:methods*; D014867:Water / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D014867:Water; D002244:Carbon; D017506:H(+)-K(+)-Exchanging ATPase
keywords: H+,K+-ATPase; cryo-electron microscopy; electron crystallography; membrane proteins; two-dimensional crystals