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 mu g/ml and pH 5 acetate buffer, we have obtained crystals that diffract to better than 15 Angstrom when prepared in negative stain,A projection map with a resolution of 30 Angstrom was calculated with unit cell dimensions of a = b = 157 Angstrom and P3 symmetry. The complex is triangular in shape with six distinct lobes observed, Additionally, six smaller structures protrude from the triangular core. (C) 1997 Academic Press.