Rotary and unidirectional metal shadowing of VAT: Localization of the substrate-binding domain

AAA-ATPases have important roles in manifold cellular processes. VAT (valos ine-containing protein-like ATPase of Thermoplasma acidophilum), a hexameri c archaeal member of this family, has the tripartite domain structure N-D1- D2 that is characteristic of many members of this family. N, the N-terminal domain of 20.5 kDa, has been implicated in substrate binding. We have appl ied rotary and unidirectional shadowing to VAT and an N-terminally deleted mutant, VAT(DeltaN), in order to map the location of this domain. For the a nalysis of data derived from unidirectionally shadowed samples we used a ne w approach combining eigenvector analysis with surface relief reconstructio n. Averages of rotary shadowed particles as well as relief reconstructions map the N-terminal domains to the periphery of the hexameric complex and re veal their bipartite structure. Thus, this method appears to be well suited to study the conformational changes that occur during the functional cycle of the protein. (C) 2000 Academic Press.