Abstract
Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.
Highlights
Cholera toxin (CT),’ produced by the Gram-negative bacterium Vibrio cholerae, is a protein of M, = 84,000 comprised of an A-subunit (MI-27,400) which contains the enzymatic activity of the toxin and a B-subunit(Mr-58,000) capable of binding a membrane receptor
The results provided projection structures at -30-A resolution for CT and -17-A resolution for CTB, which are insufficient for detailed structural analysis
Large crystals of CT hadpreviously been grown bythe vapor diffusion method at room temperature using PEG 4000 as precipitant(6)
Summary
The lyophilized toxins were dissolved in distilled water to give protein solutions in the buffer system, 0.05 M Tris, 0.2 M NaCl, 0.001 M EDTA, and 0.02% NaN3, pH = 7.5. Crystallization-CTB crystals were grownat room temperature by the vapor diffusion method using both the hanging-drop and sittingdrop procedures (7).The hanging-drop procedure utilized 1 ml of 24% (w/v) PEG 3350 containing 0.07 M Tris-HC1, 0.04 M NaCl, 0.0005 M EDTA, and 0.05%NaN3, pH = 7.75, in the well of a Linbro plastic tissue culture plate (stock no 76-033-05) as reservoir solution.
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