Abstract
Received 7 June 2013; accepted 21 June 2013Electron 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.
Highlights
Since the first report of the three-dimensional (3D) structure of the membrane protein bacteriorhodopsin [1], electron crystallography of two-dimensional (2D) crystals has become a valuable approach for determining the structure of membrane proteins [2]
In the carbon sandwich preparation, a solution containing 2D crystals is placed on a molybdenum grid that is sandwiched between two sheets of symmetric carbon films, and excess liquid is blotted away from the side of the grid with filter paper prior to freezing
Decylmaltoside (DM)-solubilized H+,K+-ATPase grown in dialysis buffer containing aluminum fluoride (AlF) produced single-layered sheet crystals, while an octaethyleneglycol dodecylether (C12E8)-solubilized preparation in berylium fluoride (BeF) with or without the specific inhibitor SCH28080 resulted in thick tubular crystals
Summary
Since the first report of the three-dimensional (3D) structure of the membrane protein bacteriorhodopsin [1], electron crystallography of two-dimensional (2D) crystals has become a valuable approach for determining the structure of membrane proteins [2]. It has been demonstrated that this preparation compensates for the image shift that causes beam-induced specimen charging, and dramatically increases the yield of good images obtained at high-tilt angles [17]. Besides its ability to compensate for the image shift, 2D crystals placed between two carbon films are expected to be better preserved in a hydrated state compared with standard single carbon support film preparations [11]. Together with its strong compensation effect against image shift due to specimen charging, which is critical when imaging tilted specimens [17], the carbon sandwich preparation technique allows the extraction of high-quality structural information from preserved 2D crystals, thereby enhancing data collection for 3D reconstruction
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