Reverse gyrase is a type IA topoisomerase, found in various hyperthermophiles and promotes ATP-dependent positive supercoiling of DNA. Electron microscopy combined with single particle analyses revealed the three-dimensional structure of the DNA-free Sulfolobus tokodaii reverse gyrase and two-dimensional average images of both the protein alone and that complexed with double-stranded DNA. The 23 Angstrom resolution map exhibited a parallelogrammatic morphology of 110 x 87 x 43 Angstrom, which is in good agreement with the crystal structure of the Archaeoglobus fulgidus reverse gyrase. The average image of the complex revealed that the monomeric enzyme binds DNA duplex. Together with this average image of the complex, the three-dimensional map implies that, at the beginning of the supercoiling reaction, DNA is bound within a 10-20 Angstrom wide cleft in the helicase-like domain. We also speculate that DNA may pass through a 20 Angstrom wide hole at the end of the cleft. (C) 2002 Elsevier Science (USA). All rights reserved.