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Sau3AI is a type II restriction endonuclease that recognizes the palindromic sequence 5'-GATC-3' and cleaves 5' to G residue on each strand. The E64A mutant full length protein was cloned and expressed in Escherichia coli. The purified (His) (6)-tagged protein has monomer and dimer fraction and was crystallized by the hanging-drop vapor-diffusion technique. The dimer protein crystals can diffract to 3.0A. resolution and the monomer protein crystals can diffract to better than 2.8A. resolution. One completed dataset has been collected and it shows that the monomer orthorhombic Sau3AI/E64A crystal is in space group C2221 with unit cell parameters (69.44, 197.60, 191.46, 90, 90, 90) and contains two molecules in one asymmetric unit.