Extended Data Figure 1. Purification and reconstitution of 3a.

(a) Size exclusion chromatogram of 3a expressed in insect cells and extracted and purified in DDM (left). Pooled fractions corresponding to dimeric 3a are highlighted in blue. Coomassie-stained SDS-PAGE of pooled dimeric 3a-containing fractions (center) and of 3a following reconstitution into PC lipids (right). This is a representative preparation used for 3a proteoliposome experiments. WT 3a was purified into DDM six times with similar biochemical behavior. Proteoliposomes from three Soy PC preparations were used for activity assays. (b) Size exclusion chromatogram of dimeric 3a reconstituted into MSP1E3D1 lipid nanodiscs (left). Pooled fractions are highlighted blue. 3a was incorporated into nanodiscs three separate times with similar biochemical behavior, and this representative preparation was used for dimeric 3a cryo-EM data collection (c,d) Same as (a,b), but for tetrameric 3a. The tetrameric peak has been observed in all WT 3a preparations. This representative preparation was used for cryo-EM of tetrameric 3a. (e) GFP fluorescence chromatogram of 3a expressed in SF9 cells and extracted in DDM detergent. Samples were run on a Superose 6 column.