Electron density projection map of the botulinum neurotoxin 900-kilodalton complex by electron crystallography

The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer two-dimensional crystallization technique. Based on the binding characteristics of the hemagglutinating portion of the complex, a number of ganglioside/ lipid mixtures were tested but only lactosyl ceramide/1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine was found to crystallize the complex. The optimum lipid mixture contained 75 mass % lactosyl ceramide and 25 mass % 1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine. Using protein concentrations from 5 to 500 micrograms/ml and pH and 5 acetate buffer, we have obtained crystals that diffract to better than 15 A when prepared in negative stain. A projection map with a resolution of 30 A was calculated with unit cell dimensions of a = b = 157 A and P3 symmetry. The complex is triangular in shape with six distinct lobes observed. Additionally, six smaller structures protrude from the triangular core.