Ring-shaped structures on KMTs in vivo. (A) MT polymerized in vitro from bovine tubulin, then mixed with recombinant Dam1 and negatively stained. (B) Negatively stained Dam1 rings. (B1) Unpublished image of the Dam1 ring (arrowhead) surrounding an MT (arrow), courtesy of E. Nogales (personal communication). (B2 and B3) Single (B2) and average of multiple (B3) negatively stained Dam1 rings in isolation (Wang et al. [2007], with permission from Nature Structural & Molecular Biology). (C) 4-nm slices cut from single KMTs and from averaged KMTs (labeled “Avg.,” then the number of MTs was averaged) from three budding yeast cells. Bottom panels are cross sections at the positions marked by black plusses in the top panels. The arrow indicates the MT wall in cross section; arrowheads indicate the ring and partial rings surrounding the MTs. (D) Comparable images for KMTs from fission yeast. (E) Localization of the S. pombe Dam1 complex, triply tagged with GFP, was accomplished with anti-GFP and a secondary anti-rabbit Fab coupled to 5 nm colloidal gold (arrowheads). The top image is a tomographic slice showing the stain and MT in the same slice. The bottom two images show a different tomogram; the top image slices the section’s surface where the antibody is bound (arrowheads), and the bottom image shows the MT in perfect alignment with the top image, so the position of the gold can be identified. Bars: (A) 100 nm; (B) 25 nm; (C and D) 50 nm; (E) 75 nm.