STRUCTURE OF TRIAD JUNCTION

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(Supported in part by NIH AR40615 to T. Wagenknecht) The frog muscle triad junction is being used as a test specimen to develop automatic procedures for generating tomograms from ice-embedded specimens. This junction contains the calcium release channel which is a macromolecular assembly that releases calcium into the t-tubes and sarcoplasmic reticulum. Our efforts are complementary to the reconstructions of the calcium release channel being in Dr. Wagenknecht's group using the single-particle approach. In the BMIRR grant proposal, the triad junction was designated as one of the three objects (the others were the mitochondrion and the primary cilium) which would be used for development and testing of the automated tomography system. Four double-tilt reconstructions of triad junctions from high-pressure frozen, freeze-substituted frog sartorius muscle were made (from a block provided by Dr. Roger Craig of U. Mass Medical School). One reconstruction was made from a 0.25lm thick section, and three from 0.125lm thick sections. Slices from the reconstructions were traced using Sterecon, and surface rendered views were created. Frozen-hydrated, plunge-frozen preparations of isolated triad junction vesicles were examined to find the optimal specimen preparation technique for cryo-tomography. It was found that a colloidal gold solution could be mixed with the vesicle suspension, which allowed us to use the gold beads for alignment markers and focusing. The first frozen-hydrated reconstruction at Albany was subsequently made with this material. Thirty tilt images were recorded from +/-600 at 4-degree intervals. The electron dose was 10 electrons/E2 for each tilt image. The pixel size of the reconstruction was 1.4nm, and the 3-D resolution was 3-4nm. One plastic-section reconstruction was shown by Dr. Rath at the Ringberg Tomography meeting in Germany, and another used in the paper resulting from the meeting. Results were also shown by M. Marko at the 10th International Conference on 3-D Microscopy in Buffalo. The cryo reconstruction was shown by C. Mannella at a presentation about the BMIRR at the MSA meeting in Cleveland. Rath, B.K., Marko, M., Radermacher, M., Frank, J. 1997 Low-dose automated electron tomography: a recent implementation. J. Structural Biology (in press.) Marko, M., Buttle, K.F., Frank, J., Khodjakov, A., Mannella, C.A., McEwen, B.F., Rath, B.K., Rieder, C.L. 1997 Electron Tomography of Cellular Organelles, a Sampling of Recent Results Cell Vision 4:(2), 137-138. (Proceedings of "Focus on Multidimensional Microscopy 1997). Frank, J., Mannella, C.A., Rieder, C. 1997) An integrated biological imaging facility: capabilities of the Biological Microscopy and Image Reconstruction Resource. Microsc. and Microanalysis 97 Ed. G. Bailey, Jones and Begell, New York, 271-272.