FIG 1.

Overall architecture of yeast NuA4. (A) Optimizing buffer conditions for purification of native NuA4 from yeast expressing C-terminally FLAG-tagged Epl1. Elutions from FLAG purification of NuA4 using the base buffer, base plus CHAPS buffer, and base plus CHAPS plus magnesium acetate (MgOAc) buffer were analyzed by SDS-PAGE, followed by silver staining. (B) Glycerol gradient ultracentrifugation purification. Elutions from the FLAG purification in the base plus CHAPS plus MgOAc buffer were subjected to glycerol gradient ultracentrifugation, and individual fractions from the gradient were analyzed by SDS-PAGE. The fraction indicated by an asterisk was used for further EM analysis. (C) Representative segment of a negative-stain EM raw image of purified NuA4. Scale bar, 100 nm. (D) 2D class averages obtained for NuA4 purified from anti-FLAG affinity chromatography and gradient fixation (GraFix) using the base buffer with or without the addition of CHAPS and MgOAc. The two sets of averages (base buffer, modified buffer) were calculated from 9,869 and 23,165 particles, respectively. The box edge length is approximately 467 Å. (E) 2D class averages NuA4 in different orientations. The box edge length is approximately 467 Å.