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.

Keywords: H+,K+-ATPase; cryo-electron microscopy; electron crystallography; membrane proteins; two-dimensional crystals.

Fig. 1.

Negatively stained 2D crystals of H⁺,K⁺-ATPase at different conformations show the variety in their morphologies. Flat-sheet crystals of E2AlF (a) consist of a single-crystalline array, shown as a single reciprocal lattice in its Fourier transformation (b, indicated as a* and b*). On the other hand, flattened tubular crystals of (SCH)E2BeF (c) and vesicular crystals of (Rb⁺)E2AlF (e), and their Fourier transformations (d and f, respectively) show two overlapping reciprocal lattices (a₁*, b₁* and a₂*, b₂*). Thus, each layer of these crystals was processed independently as two overlapping crystalline layers. Scale bars for crystal images and their Fourier transforms are shown in panel E (2 μm) and panel F (1/50 Å⁻¹), respectively. (g) Crystal packing of (SCH)E2BeF crystals shows that the inter-molecular contact between the N-terminal tail of β-subunit and the N domain (arrowhead), and the protruded structure of the P domain and the outermost portion of the A domain (double arrowhead) at the cytoplasmic side of the molecule. Color surface indicates EM map of H⁺,K⁺-ATPase αβ-protomer (blue, A domain; yellow, N domain; green, P domain; light gray, TM helices; pink, β-subunit) with a superimposed ribbon model. Green mesh indicates symmetry-related neighboring molecules and wheat-colored boxes indicate approximate locations of lipid bilayers. Due to crystal packing, the single-crystalline layer (indicated by a grey bar) consists of two lipid bilayers (indicated as wheat-colored boxes), which is responsible for the one-crystalline layer of each crystal in the different morphologies (red dotted boxes in lower panel cartoon). For interpretation of colour in this figure, the reader is referred to the web version of this article.

Fig. 2.

Comparison of the analyzed image quality between carbon sandwich and single carbon support film preparations. (a–c) Low magnification images (search mode) of H⁺,K⁺-ATPase 2D crystals (arrowheads) in frozen specimens prepared by the single carbon method (a and b) or carbon sandwich method (c). Most of the crystals embedded in the thin ice are dehydrated in the single carbon preparation (a), while preserved crystals can be found in the thicker ice area at the edge of the grid well (*: dark area) in some cases (b). In the carbon sandwich preparation, preserved 2D crystals are distributed evenly over the grid well (c). White arrows indicate the position used for focusing the image, and black arrows indicate crinkling of the carbon membrane, which usually occurs in carbon sandwich preparations. Because all images herein were obtained using a low-dose defocused diffraction mode, the mean diameter of ∼2 μm for sheet crystals is used for approximate scaling. (d and e) IQ-plot calculated from a non-tilted image of H⁺,K⁺-ATPase single sheet crystal in the E2AlF conformation prepared by the single carbon support film (d) and carbon sandwich (e) techniques.

Fig. 3.

Cartoons depicting the cross section of the specimen prepared by single carbon support film (a) and that by carbon sandwich technique (b). Black lines indicate carbon support films, gray boxes indicate the molybdenum grid and light blue indicates the embedding buffer. As water evaporated (tan wavy arrows) from the surface of the specimen (a), most of the crystals were broken by dehydration (grey dotted line). Preserved crystals (green dotted line) were always observed in the thick ice at the edge of the grid (Fig. 2b), while the resolution is limited (Fig. 2d) due to the increased concentration of the embedding buffer by dehydration (dark blue). In contrast, water evaporation occurred only at the blotting position where the upper carbon membrane was partially broken by filter paper in the carbon-sandwiched specimen (b). Thus, the concentration change of the embedding buffer was limited around 2D crystals sandwiched between two carbon films (Fig. 2c), while the salt concentration was increased at the blotting position (b). Removal of excess water by blotting or evaporation induced spontaneous water flow (dotted arrow), which may prevent free diffusion of concentrated reagents to the central area of the grid. Therefore, the microenvironment of the 2D crystals embedded in the thin water layer seems to remain constant during preparation, and thus the inherent crystal qualities are preserved in the carbon sandwich preparations (Fig. 2e). For interpretation of colour in this figure, the reader is referred to the web version of this article.

Fig. 4.

Representatives of Fourier transforms of a crystal image taken from non-tilted (a), 45° (b) and 70° (c) tilted specimens prepared by the carbon sandwich technique, show isotropic diffraction spots as well as Thon rings and are thus well suited for image processing [19]. In contrast, spots just along the tilt axis (dotted lines) are visible in the Fourier transform of 20° tilted crystals from single carbon preparation due to beam-induced specimen charging (d). Reciprocal lattice vectors (a*, b*) are indicated as arrows, and tilted axes are shown as dotted lines in tilted data. A scale bar is shown in panel D (1/50 Å⁻¹).

Fig. 5.

Statistics of data collection from carbon-sandwiched specimens. Success rate was calculated from the number of micrographs merged into the 3D structure factor per total number of micrographs obtained, for each crystal sample in a different conformation of the transport cycle as indicated by different colors in the figure. For interpretation of colour in this figure, the reader is referred to the web version of this article.