The use of synchrotron radiation has been essential in the study of Botulinum neurotoxin. Crystals of type A that diffract weakly """"""""in-house"""""""" to 4.5 E diffract strongly to 3.3 E on both SSRL beamlines 7-1 and 9-1. Using these data we were able to solve the structure of the native toxin using MIR methods and refine the model to Rw=20.0% and Rfree=27.9%. We are asking to extend our proposal to allow for the study of the toxin in complex with inhibitors, an activator, and its ganglioside receptor. We feel these projects will be more straightforward due to new freezing conditions for cryo-crystallography and the ability to use molecular replacement. In addition, we would like to study the toxin in its low pH, membrane-spanning state. Using trial beam time at 7-1 in March of this year, we tested a different serotype, Botulinum neurotoxin type E. The crystals diffracted to 2.8 E with good freezing conditions. Unfortunately, a number of the crystals are twinned - a problem which is hard to identify on """"""""in-house"""""""" equipment. The use of synchrotron radiation allows for a rapid screening of suitable crystals and again improves the resolution. This new protein has high sequence homology with type A and should be solvable by molecular replacement.
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