ELECTRON CRYSTALLOGRAPHY OF NEUROTOXINS

我们已经开发了一种程序来准备高度有序的两个- 适用于电子的神经毒素的三维晶体 晶体分析。这种方法涉及到形成 一种含有水中特定配体的脂单分子层：空气 界面。我们已经成功地制备了二维晶体 几种毒素和神经节苷脂结合片段。光学 负染的电子显微照片的衍射图 晶体包括大约20到25埃的反射。 决议。 对于天然滤液毒素，三维密度图在 重建了大约20埃的分辨率，揭示了 分子具有不对称的三叶结构。对于 蛋白酶裂解毒素，含显微照片的倾斜系列 已经记录了+60度到-60度之间的倾斜角 一张分辨率为20埃的三维密度图 都被重建了。这表明分子本质上是“V”型的。 很有型。V的碱基被认为含有受体 结合部位。 这项提议是为了延长这项工作。我们将研究 以改善为目标的晶体形成条件 结晶过程和晶体的质量。 我们将使用未染色的方法检测天然滤液破伤风毒素 水合制剂。这可能会提高分辨率和 其他结构详图。我们的长期计划是延长 这些分析包括单肽细胞提取物形式的 破伤风毒素、一些肉毒杆菌神经毒素以及 这些毒素的神经节苷脂结合肽。决心 中的主要分子结构域。 这些有趣的神经毒素将提供关于 分子与细胞膜之间的相互作用。我们的龙 范围研究将使我们能够比较这些结构 非常相似的神经毒素。 这些都是不同寻常的重要标本，应该 特别适合用电子显微镜检查 技巧。这项技术应该适用于 一种新的二维类晶体的制备方法 各种生物标本。

We have developed a procedure for preparing highly ordered two- dimensional crystals of neurotoxins suitable for electron crystallographic analysis. This method involves the formation of a lipid monolayer containing a specific ligand in the water:air interface. We have successfully prepared two-dimensional crystals of several toxins and a ganglioside binding fragment. Optical diffractograms of electron micrographs of negatively stained crystals include reflections to about 20 to 25 angstroms resolution. For native filtrate toxin the three dimensional density map at about 20 angstroms resolution was reconstructed and reveals the molecule to have an asymmetric three-lobed structure. For the protease cleaved toxin, tilt series containing micrographs with tilt angles between +60 degrees and -60 degrees have been recorded and a three dimensional density map at 20 angstroms resolution has been reconstructed. This shows the molecule to be essentially "V" shaped. The base of the V is proposed to contain the receptor binding site. This proposal is to extend this work. We will examine the conditions for forming crystals with the objectives of improving the process of crystallization and the quality of the crystals. We will examine native filtrate tetanus toxin using unstained hydrated preparations. This may allow improved resolution and additional structural detail. Our long range plans are to extend these analyses to include the single peptide cell extract form of tetanus toxin, some of the botulinum neurotoxins as well as the ganglioside binding peptides of these toxins. The determination of these structures will reveal the major molecular domains in these interesting neurotoxins and will provide information about the interaction between the molecules and cell membranes. Our long range studies would allow us to compare the structures of these very similar neurotoxins. These are unusual and important specimens which should be especially well suited for examination using electron microscopic techniques. This technology should be applicable to the preparation of similar two-dimensional crystals prepared from a variety of biological specimens.