Windex: a toolset for indexing helices Andrew Ward, a Michael F. Moody, b Brian Sheehan, a Ronald A. Milligan, a and Bridget Carragher a, * a Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA b School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK Received 2 September 2003, and in revised form 6 October 2003 Abstract We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen. Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three- dimensional reconstruction of the macromolecular structure. The process of helical reconstruction has been largely automated except for the critical first step of indexing the helical diffraction pattern. This is quite often the rate-limiting step in the overall process, particularly in the case of large helical tubes, which have complicated helical diffraction patterns that may vary from tube to tube. We have developed a set of procedures, supported by a graphical user interface, that provide a straightforward and semi-automated approach to indexing a helical structure. The new procedures have been tested using a number of helical specimens, including TMV, acto-myosin, decorated microtubules, and a variety of helical tubes of a bacterial membrane protein. Ó 2003 Elsevier Inc. All rights reserved. 1. Introduction There are a number of advantages to determining macromolecular structure from helical filaments or tubes. The order imposed by the helical symmetry allows for some quality assessment of each image and pre- sumably also helps to some extent in immobilizing each macromolecule in a fixed conformational state, which may be important for reaching high resolution. Two recent publications have demonstrated that helices can be used to reconstruct electron density maps to a reso- lution that allows for interpretation of atomic structure (Miyazawa et al., 2003; Yonekura et al., 2003). Al- though structures arranged as two-dimensional (2D) crystals also share these advantages, with helices it is not necessary to acquire images of the tilted specimen, which is often a rate-limiting step in acquiring a complete da- taset from 2D crystals. Most of the processing steps for reconstructing he- lical structures are fairly straightforward and a number of software packages have been described in the liter- ature (Carragher et al., 1996; DeRosier and Moore, 1970; DeRosier et al., 1999; Egelman, 2000; Morgan and DeRosier, 1992; Owen et al., 1996; Toyoshima, 2000; Whittaker et al., 1995). Many of these also provide fairly extensive documentation for guiding a new user through the process of helical reconstruction. However, almost all of these packages assume that the helical diffraction pattern has already been indexed, i.e., that the pattern has been analyzed and the helical symmetry has been determined. While a number of publications describe helical diffraction theory (Cochran et al., 1952; Klug et al., 1958) and there are tutorials on the overall process of indexing helical diffraction patterns (Moody, 1990; Toyoshima, 2000), this nevertheless remains as a fairly daunting task when examining any new helical structure. An added com- plication in the case of large diameter helical tubes is that every tube may potentially have a different helical symmetry, requiring that this indexing be performed for every image. We have thus developed a set of programs, supported by a graphical user interface, which can be used as an aid in indexing a new helical diffraction pattern. The overall package, which we have called Windex, relies * Corresponding author. Fax: 1-858-784-9090. E-mail address: bcarr@scripps.edu (B. Carragher). 1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.10.008 Journal of Structural Biology 144 (2003) 172–183 Journal of Structural Biology www.elsevier.com/locate/yjsbi