Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Go1

A modified isolation procedure provides a homogeneous A(1)- ATPase from the archaeon Methanosarcina mazei Go1, containing the five subunits in stoichiometric amounts of A(3): B-3: C: D: F. A(1) obtained in this way was characterized by three- dimensional electron microscopy of single particles, resulting in the first three- dimensional reconstruction of an A(1)- ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B-3. At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A- B- A triplet. To investigate further the topology of the stalk subunits C - F in A(1), cross- linking has been carried out by using dithiobis[ sulfosuccinimidyl-propionate] ( DSP) and 1- ethyl- 3-( dimethylaminopropyl)carbodiimide ( EDC). In experiments where DSP was added the cross- linked products B- F, A(x)- D, A- B- D, and A(x)- B-x- D were formed. Subunits B- F, A- D, A- B- D, and A- B- C- D could be cross- linked by EDC. The subunit- subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three- dimensional organization of this A(1) complex is discussed in terms of the relationship to the F-1- and V-1- ATPases at a resolution of 3.2 nm.