Tetrameric KcsA incorporates efficiently into NABBs. (A) Gel filtration of empty NABBs and 1K-NABB and 4K-NABB samples. Solid (280-nm) and dashed (460-nm) curves represent protein and NBD lipid absorbance, respectively. (Inset) Coomassie-stained SDS-PAGE of gel filtration–purified samples of 4K-NABB, 1K-NABB, and KcsA in PE/PG vesicles, with (Δ) or without (−) heating at 95°C before loading. The bands represent KcsA tetramer (68 kD), monomer (17 kD), and Zap1 (31 kD). (B) Negative-stain EM image of 4K-NABBs. Some particles are seen characteristically “stacking” together, oriented with the plane of the disc and perpendicular to EM grid. Bar, 100 nm. (C) Class averages of single particles picked from EM micrographs of 4K-NABBs (panels 1–4) show density within the discs oriented at different angles relative to the plane of the disc. Cartoon representations below each class average show the likely orientation of the densities emerging from the discs. Class averages of empty NABBs (panels 5 and 6) lack these densities. Each class average was computed from ∼200 isolated single particles. The side of each panel is 30 nm.