CHAPERONIN FILAMENTS ARCHAEAL CYTOSTRUCTURE

（由DOE 61225-00-110向J. Trent提供部分支持） 当今生物学的基本问题是蛋白质如何折叠成它们的 在拥挤的活细胞中发挥作用。 最近 研究表明，这一过程是由一类蛋白质介导的 叫做分子伴侣 最重要的监护人之一， 这个过程是一个多亚基复合物，称为伴侣蛋白， 一种双环结构，被认为是在其中空部分折叠蛋白质， 核心 我们从纯化的伴侣蛋白中得到的证据表明 在体外形成有序的细丝，并在细胞中观察到， 表明在体内存在类似细丝。 我们建议采用基准利率 设施1。 查看最低限度处理的冷冻水合细胞内部 或通过HVEM或IVEM进行厚冷冻切片，以改善 通过常规EM技术在细胞中看到的内部结构; 2. 收集负染色和冷冻水合的高质量图像 与细胞中看到的细丝相比， 澄清它们的结构关系，并深入了解 伴侣蛋白丝形成的基础;和3. 使用3D 断层重建设备， 他们试图理解这种结构的性质， 用于附着其他细胞成分的位点。 在特伦特医生来访期间， 我们检查了冷冻切片（Tokuyasu方法）S.柴田细胞 HVEM，并制作了新鲜制备的细胞整装制剂 在文化中成长。 录制了许多立体声对， 从切片进行断层重建。 的 重建显示，S层是完整的， 醋酸盐染色剂没有进入细胞内。 在整个山脉中，我们 可以看穿整个细胞并看到染色的S层， 但我们看不到内部结构。 我们还研究了 培养的细胞与视频显微镜。 不规则的豆袋状的形状 这可能是由于内部细胞骨架或细胞骨架的特征。 S层和细胞的膨压。 特伦特博士还记录了 几对立体细胞， 希望看到伴侣蛋白的结构以及它的 架构 这些图像非常难以解释。 样品 特伦特博士带来的大肠杆菌（L型），以及活细胞， 环境和高温条件下， 压力冷冻总共30次。 将细胞 冷冻取代和包埋。 这些积木被送到特伦特医生那里 切片

(Supported in part by DOE 61225-00-110 to J. Trent) One of the fundamental problems in biology today is how proteins fold into their functioning form in the crowded confines of living cells. Recent studied suggest that this process is mediated by a class of proteins called molecular chaperones. One of the most important chaperones in this process is a multi-subunit complex known a chaperonin that is a double-ring structure believed to fold proteins inside its hollow core. We have evidence from purified chaperonins that they aggregate in vitro to form ordered filaments, and observations in cells that suggest similar filaments exist in vivo. We propose to use the BMIRR facilities to 1. Look inside minimally treated frozen hydrated cells or thick cryosections by HVEM or IVEM to improve the images of the internal structures seen in cells by conventional EM techniques; 2. Collect high quality images of negatively stained and frozen hydrated chaperonin filaments to compare to the filaments seen in cells to clarify their structural relationship and to gain insights into the basis for chaperonin filament formation; and 3. Use the 3D tomographic reconstruction facilities to image the filaments themselves in an attempt to understand the nature of this structure as a site for attaching other cell components. During Dr. Trent's visit, we examined cryosectioned (Tokuyasu method) S. shibatae cells with the HVEM, and made fresh-prepared whole-mount preparations of cells growing in culture. Many stereo pairs were recorded, and a tomographic reconstruction was made from a section. The reconstruction revealed that the S-layer is intact and that uranyl acetate stain did not get inside the cell. In the whole mounts, we could see through the whole cell and visualize the stained S-layers, but we could not see the internal structures. We also looked at the cultured cells with video microscopy. The irregular, bean-bag, shape seen may be due to an internal cytoskeleton or to features of the s-layer and the cell's turgor pressure. Dr Trent also recorded several stereo pairs of cells that had the surface layer removed in hopes of seeing the structure of chaperonin as well as its architecture. These images were very difficult to interpret. Samples of E-coli (L-forms), brought by Dr. Trent, as well as live cells at both ambient and high-temperature conditions, were high pressure-frozen for a totoal of 30 runs. The cells were freeze-substituted and embedded. The blocks were sent to Dr. Trent for sectioning.