The yeast mitochondrial ATPase has been genetically modified to include a HiS(6) Ni-affinity tag on the amino end of the mature P-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F-1-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 Angstrom resolution. (C) 2004 Elsevier Inc. All rights reserved.