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T.G. Wensel, F. He, Z. Zhang; Cryo–Electron Microscopy and 3D Reconstruction of PDE6 and Its Complexes With Membranes and Transducin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1181.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the structures of different conformations of photoreceptor cGMP phosphodiesterase, PDE6, and its complexes with membranes and active transducin. Methods: Cryo–electron microscopy was carried out on samples of purified PDE6 in the presence and absence of activated purified transducin (Gtα–GTPγS) and of phospholipid vesicles or monolayers. Samples were imaged either unstained in vitreous ice or in uranyl acetate negative stain. Images were processed using EMAN software for 3D reconstruction by single particle analysis. In the iterative refinement process, the "multirefine" option was selected to identify individual particles (images of molecules) falling into one of two distinguishable structural classes. Results: The overall shape of PDE6 observed in both ice and negative stain is similar to the bell–shaped structure observed previously by EM of negatively stained samples [Kajimura N. et al., .J Struct Biol. 2002; Kameni Tcheudji JF et al., J. Mol. Biol. 2001]. Reconstructions were generated of two distinct conformations. In ice PDE6 is preferentially oriented with its faces of largest area perpendicular to the grid surface, but this distribution is altered in the presence of activated transducin. PDE6 bound to vesicles could be observed in ice and was observed much more frequently in the presence of activated transducin; the bottom of the "bell" is the membrane attachment surface. Activated transducin and PDE formed arrays on the surface of tubular vesicles, in a manner reminiscent of helical crystals of holo–transducin (Gtαßγ–GDP) [Zhang, Z. et al., J. Biol. Chem., 2004], but tubes formed by PDE and lipids in the absence of transducin were much more poorly ordered. Conclusions: If PDE6 is approximated as a flattened bell, the base of the bell is the membrane–binding surface, presumably the site of its isoprenylated carboxyl–termini. The same binding motif is seen when it is bound to activated transducin, and these membrane–bound assemblies should allow determination of the structure of the G protein–effector complex.
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