ELECTRON CRYSTALLOGRAPHY OF NEUROTOXINS

我们开发了一种制备高度有序的双- 适合电子的神经毒素的三维晶体 晶体学分析 该方法包括形成 在水：空气中含有特定配体的脂质单层 接口. 我们已经成功地制备了二维晶体 几种毒素和一种神经节苷脂结合片段 光学 负染色的电子显微照片的衍射图 晶体包括约20至25埃的反射 分辨率 对于天然滤液毒素， 大约20埃的分辨率被重建，并揭示了 分子具有不对称的三叶结构。 为 蛋白酶裂解毒素，倾斜系列包含显微照片， 已经记录了+60度和-60度之间的倾斜角度 一个20埃分辨率的三维密度图 被重建。 这表明分子基本上是"V"型的 形状。 V的底部被提议包含受体 结合位点 这项建议是为了扩大这项工作。 我们会研究 形成晶体的条件，目的是改善 结晶的过程和晶体的质量。 我们将使用未染色的 水合制剂。 这可以允许改进的分辨率， 更多结构细节 我们的长期计划是 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。 测定 这些结构将揭示主要的分子结构域， 这些有趣的神经毒素，并将提供信息， 分子和细胞膜之间的相互作用。 我们漫长 范围研究将使我们能够比较这些 非常相似的神经毒素 这些都是不寻常的重要标本， 特别适合于使用电子显微镜检查 技术. 这项技术应该适用于 制备类似的二维晶体， 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.