The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IICglc) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IICglc was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IICglc into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IICglc, which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6 angstrom and 4 angstrom, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93 angstrom resolution. These 2D and 3D crystals of wt-IICglc lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain. (C) 2015 Elsevier Inc. All rights reserved.