Helicobacter pylori secretes a vacuolating toxin (VacA) that can assemble into water-soluble oligomeric complexes and insert into membranes to form anion-selective channels. Previous studies have described multiple types of oligomeric VacA structures, including single-layered astral arrays, bilayered forms, and two-dimensional crystalline arrays. In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified and analyzed, and three-dimensional models of the bilayered forms of VacA were constructed with a resolution of about 19 angstroms. These bilayered forms of VacA have a "flower"-like structure, consisting of a central ring surrounded by symmetrically arranged peripheral "petals." Further structural insights were obtained by analyzing a mutant form of VacA (VacA Delta 6-27), which lacks a unique amino-terminal hydrophobic segment and is defective in the capacity to form membrane channels. Bilayered oligomeric complexes formed by wild-type VacA contained a visible density within the central ring, whereas bilayered complexes formed by VacA Delta 6-27 lacked this density. These results indicate that deletion of the VacA amino-terminal hydrophobic region causes a structural alteration in the central ring within VacA oligomers, and suggest that the central ring plays an important role in the process by which VacA forms membrane channels. (C) 2005 Elsevier Inc. All rights reserved.