The (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS-PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75 angstrom resolution. The crystal belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 82.0 angstrom, c = 139.8 angstrom, alpha = beta = gamma = 90.0 degrees. (C) 2015 The Authors. Published by Elsevier Inc.