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ELECTRON CRYSTALLOGRAPHY OF NEUROTOXINS

神经毒素的电子晶体学

基本信息

批准号：
3300787
负责人：
GERALD J STUBBS
金额：
$ 8.23万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1994-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3300787
关键词：
X ray crystallography chemical binding chemical stability crystallization electron density electron microscopy gangliosides laboratory mouse neurotoxins protein structure receptor tetanus toxin

项目摘要

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.

我们开发了一种制备高序二-的方法