CDCs and MACPFs, structures and mechanism. (A) Crystal structures of perfringolysin (Rossjohn et al., 1997), the Photorhabdus luminescens MACPF. (Rosado ...
CDCs and MACPFs, structures and mechanism (A) Crystal structures of perfringolysin (Rossjohn et al., 1997), the Photorhabdus luminescens MACPF (Rosado et al., 2007) and C8α (Hadders et al., 2007) MACPF, with equivalent regions of the structures coloured the same. (B) Homology model of domains within PFN, constructed using the Phyre webserver (http://www.sbg.bio.ic.ac.uk/~phyre/) (Kelley et al., 2000; Bennett-Lovsey et al., 2008): MACPF domain red, EGF-like domain green and C2 domain blue. Regions missing from the structures are dotted. (C) CryoEM reconstructions of the prepore (left) and pore (right) states of the CDC pneumolysin (Tilley et al., 2005). The domains of perfringolysin have been fitted within one subunit in each (see D). (D) Fits of the domains of perfringolysin to the prepore (left) and pore (right) states of pneumolysin. Domains 1 and 3 of perfringolysin (encompassing the MACPF-like domain of the protein) are coloured red, domain 2 green and domain 4 blue, to highlight similarities to PFN. In the pore state domain 3 is obviously significantly restructured since it does not fit the density well. (E) As defined using fluorescence studies (Shatursky et al., 1999; Heuck et al., 2000, 2003), domain 3 refolds on pore formation to create a β-stranded wall surrounding the pore. The regions that refold consist of six α helices (coloured red in panel A). The complete pre-pore oligomer and pore oligomer fits are shown in red, and a single refolded pore state subunit on the right (Tilley et al., 2005).
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The effect of variable pore diameter on the appearance of an average of pore images To show the effect of variable pore diameter we took the one-sided pore-forming state class average (see main paper, Figures 6D and 6F) and applied a range of different translation shifts +1 to +20 pixels to its mirror. Adding the original pore state class average and each mirror image together generated the 20 images shown above, a simulated set of pores of differing diameters. The last image displayed is the average of the preceding 20 and shows how a variable size to the pore results in the smearing of one side of the structure on class average computation, if the images have been aligned with respect to one side of the structure only. This simulates the effect of such a one-sided alignment. These images were generated using IMAGIC (van Heel et al., 1996).
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