STRUCTURE OF TRIAD JUNCTION

(部分由NIH AR40615到T.Wagenkeck支持)青蛙 肌三联会正在被用作测试样本，以开发 从冰层中生成层析图像的自动过程 标本。该连接含有钙释放通道，该通道 是一种将钙释放到t型管中的大分子集合体。 和肌浆网。我们的努力是对 DR患者钙释放通道的重建 瓦根克内赫特的小组使用单粒子方法。在BMIRR中 批地建议，三合会交界处被指定为三个 对象(其他为线粒体和初级纤毛) 将用于开发和测试自动化的 断层扫描系统。三合会的四次双倾斜重建 高压冷冻、冷冻替代的青蛙缝纫肌的连接 肌肉是由马萨诸塞州大学的罗杰·克雷格博士提供的块 医学院)。其中一个重建是从0.25lm厚的 截面，以及三个0.125 lm厚的截面。切片来自 使用Sterecon跟踪重建，并使用表面渲染视图 都被创造出来了。冷冻-水合-急速冷冻制剂 检查三联体连接小泡以找到最佳标本 低温层析成像的制备技术。结果发现，一种 胶体金溶液可以与囊泡悬浮液混合， 这使得我们可以使用金珠作为对准标记 集中注意力。奥尔巴尼的第一个冷冻水化重建是 后来用这种材料制成的。30张倾斜图像 以4度间隔从+/-600开始记录。电子剂量为10 每个倾斜图像的电子数/E_2。对象的像素大小。 重建波长为1.4 nm，三维分辨率为3~4 nm。一 Rath博士在Ringberg展示了塑料切片重建 在德国举行的层析成像会议，以及在论文中使用的另一种结果 从会议上。M·马尔科在10日也展示了结果。 在布法罗举行的国际三维显微镜会议。冷冻机 重建由C.Mannella在一次关于 BMIRR在克利夫兰举行的MSA会议上。Rath，B.K.，Marko，M. 雷德马赫，M.，弗兰克，1997年低剂量自动电子学 体层摄影术：最近的一种实现。J.结构生物学(In 按下。)首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容 王永明，陈晓华，等.电子断层扫描技术 细胞器，细胞视觉的最新结果样本 4：(2)，137-138。《聚焦多维显微镜》论文集 1997年)。Frank，J.，Mannella，C.A.，Rieder，C.1997) 生物成像设备：生物的能力 显微镜和图像重建资源。Microsc.和 微量分析97版。G.贝利，琼斯和贝格尔，纽约，271-272。

(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.