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.
| Flicker, P F; Robinson, J P; DasGupta, B R (1999) Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide? J Struct Biol 128:297-304 |
