Purification and imaging of the (PA63)7–(LF)3 complex. (A) Superose 6 gel filtration shows a peak for the (PA63)7 heptamer that shifts to higher mobility (larger size) when incubated with increasing ratios of LF. At a (PA63)7/LF molar ratio of 1:3, saturation is somewhat <100% complete, owing to the low concentrations used. At 1:4 and 1:5 ratios, saturation is complete, and the major peak is consistent with a molecular ratio of 3:7 (LF/PA63) (the peaks in the low molecular weight region comprise excess uncomplexed LF). (B) Coomassie-stained SDS-PAGE gel of purified (PA63)7–(LF)3 used for vitreous ice cryo-EM. Densitometry confirms a PA63/LF ratio of 7:3. Molecular mass is indicated in kilodaltons. (C and D) Vitreous ice cryo-EM images of (PA63)7–(LF)3 complexes collected without (C) or with (D) pre-applying a layer of carbon to the EM grid in order to have the complexes adopting multiple orientations. (E and F) (PA63)7–(LF)3 complexes shown in two characteristic orientations, side view (E) and top view (F). The first column shows reprojection of the (PA63)7–(LF)3 reconstruction; second column, the corresponding 2-D class averages corresponding to the same orientation; third through seventh columns, examples of individual complexes (PA63)7–(LF)3 in the corresponding orientations. For viewing purposes, the contrast is inverted in the projections and averages; the raw images were low pass filtered to 40 Å.
