The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly N-15-labeled protein: it comprises residues between Ile-4 and Gln-181, A 185-residue fragment, termed epsilon>(*) over bar * (1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn2+ at pH 5.8, The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degreesC, and diffract X-rays to 2.0 Angstrom. The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 Angstrom, c = 111.4 Angstrom. (C) 2000 Academic Press.