title: Rotary and unidirectional metal shadowing of VAT: localization of the substrate-binding domain.
abstract: AAA-ATPases have important roles in manifold cellular processes. VAT (valosine-containing protein-like ATPase of Thermoplasma acidophilum), a hexameric 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 applied rotary and unidirectional shadowing to VAT and an N-terminally deleted mutant, VAT(Delta N), in order to map the location of this domain. For the analysis of data derived from unidirectionally shadowed samples we used a new approach combining eigenvector analysis with surface relief reconstruction. Averages of rotary shadowed particles as well as relief reconstructions map the N-terminal domains to the periphery of the hexameric complex and reveal 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.
authors.affiliation: Abteilung Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Rockel
authors.forename: R
authors.initials: R
authors.lastname: Guckenberger
authors.forename: H
authors.initials: H
authors.lastname: Gross
authors.forename: P
authors.initials: P
authors.lastname: Tittmann
authors.forename: W
authors.initials: W
authors.lastname: Baumeister
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D016626:Shadowing Technique, Histology / Q000379:methods*; D013822:Thermoplasma / Q000201:enzymology; D000074405:Valosin Containing Protein
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein