Crystallization of isoelectrically homogeneous cholera toxin.
Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.
Spangler
B D
BD
Biological, Environmental, and Medical Research Division, Argonne National Laboratory, Illinois 60439.
Westbrook
E M
EM
D002772:Cholera Toxin / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D014961:X-Ray Diffraction
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D002772:Cholera Toxin