Fig. 4: Na⁺ is important for functional assembly of ATP-BsKtrAB.

a The SEC profiles of BsKtrA (apo BsKtrA, ATP-BsKtrA, apo BsKtrA_E125Q or ATP-BsKtrA_E125Q) mixed with BsKtrB in Na⁺ Buffer or K⁺ Buffer with 0.03% DDM. The retention volume for the BsKtrAB complex is benchmarked as the black arrow, while the retention volumes for BsKtrA octamer and BsKtrB dimer are benchmarked as the gray and white arrows, respectively. b Fluorescence-based K⁺ flux assays using BsKtrB, ADP-BsKtrAB, and ATP-BsKtrAB in Swelling Na⁺ Buffer (Na⁺, left panel) or Swelling K⁺ Buffer (K⁺, right panel). The addition of H⁺ ionophore CCCP and K⁺ ionophore valinomycin are indicated as black and white arrows. K⁺ flux rate constants were calculated by fitting the data (100–500 s) to a one-phase decay model using GraphPad Prism. c K⁺ flux rate constants (left panel) and normalized flux rate constants (right panel) of BsKtrB, ATP-BsKtrAB and ADP-BsKtrAB in the presence of Na⁺ (blue bars) and K⁺ (green bars). The normalized K⁺ flux rate constants were calculated using the respective K⁺ flux rates of BsKtrB in the presence of either Na⁺ or K⁺ as 100%. Data represent mean ± s.d. with n = 4 (for ADP-BsKtrAB in Na⁺) or n = 5 (for the others) independent experimental replicates. Statistical analyses were performed using two-way ANOVA. The schematic illustration of normalization and analysis are shown in Supplementary Fig. 15. Source data for a–c are provided as a Source Data file.