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1-Cx263T was performed as described in (22), with the following modifications. The sleep-inducing lipid oleamide (34), which has been shown to induce gap junction closure in vivo (35), and the trifluoromethylketone inhibitor of fatty acid amide hydrolase (36) were present during the induction period of the BHK cells at concentrations of 100 and 1 μM, respectively. Oleamide was added in an attempt to investigate if it caused conformational changes in comparison to our previous projection map. The addition of the inhibitor was necessary because BHK cells showed an oleamide-hydrolyzing enzymatic activity. Because the in vivo effects of the oleamide are reversible, this lipid was also added during all stages of the specimen preparation (100 μM), the detergent extraction of the membranes (200 μM), and the final dialysis (100 μM) of the sample. Stock solutions of oleamide and the trifluoromethylketone inhibitor were prepared in ethanol and added to the growth medium just before use. The final ethanol concentration in the medium was 0.1%.
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Carbon-coated molybdenum grids and frozen and hydrated specimens were prepared as previously described (22). Low-dose electron micrographs of specimens tilted from 0° to 35° were recorded on a Philips CM200FEG microscope at an accelerating voltage of 200 kV, a nominal magnification of 50,000, and defocus values ranging from -2000 to -15,000 Å. Crystal quality was assessed by the optical diffraction of electron micrographs, and only those images with spots visible to a resolution of at least 11 Å were selected for further processing. Micrographs were digitized on a Perkin-Elmer flatbed microdensitometer at a 10-μm step size (corresponding to 2 Å sampling at the level of the specimen). The MRC image-processing package, which was developed by R. Henderson and colleagues (37-39), was used to analyze the images and to correct for lattice distortions and effects of the contrast transfer function. Initial estimates for the tilt parameter of each crystal were obtained from the changes of the contrast transfer function across the image. Image data were then merged in plane group p6, and lattice lines were fitted with a real-space envelope of 220 Å. Considering only data to a resolution of 15 Å, we refined the tilt geometries of the crystals against a raw 3D model before refining the underfocus values of all images to a resolution of ∼7 Å. All images were then processed a second time with back projections of the refined 3D model for the cross-correlation search [R. Henderson, program: MAKETRAN (unpublished program)]. This gave a better description of the lattice distortions and, consequently, a more accurate correction of the image. The improved raw data were subjected to two more cycles of tilt geometry and underfocus refinements, and individual resolution cutoffs were determined for each image before the final merging of data. Density maps were calculated with and without imposing an inverse B factor to ensure that the enhancement of the weak image-derived amplitudes at higher resolution did not introduce spurious density outside the low-resolution molecular boundary. An inverse temperature factor of B = -350 was found to be adequate. Application of this sharpening increased the amplitudes at 7.5 Å by a factor of 4.7. The 3D density map was visualized with the program O (40).
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We gratefully acknowledge the technical assistance of D. W. Entrikin. This work has been supported by grants from NIH (M.Y. and N.B.G.), the Lucille P. Markey Charitable Trust (N.B.G.), the Gustavus and Louise Pfeiffer Research Foundation (M.Y.), and the Baxter Research Foundation (M.Y.), V.M.U. was a recipient of a postdoctoral fellowship from the American Heart Association. During the course of this work, M.Y. was an Established Investigator of the American Heart Association and Bristol-Myers Squibb and is now the recipient of a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund.
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