The NBD1–TMD2 (N1–T2) linker revealed in the quatrefoil-like conformation of Glib/ATP bound Kir6.1/SUR2B vascular K _ATP channels. (A) Cryo-EM map of (Kir6.1)₄-SUR2B in propeller-like conformation (EMD-23864, filtered to 5 Å and contoured to 0.7 σ) with the density of one Kir6.1 subunit and the SUR2B N1–T2 linker shown in blue and green, respectively, in side and top views. (B) Same as A but in quatrefoil-like conformation (EMD-23880, filtered to 5 Å, and contoured to 0.5 σ). (C) Bottom view of the (Kir6.1)₄-SUR2B structure in the quatrefoil-like conformation (PDB ID 7MJO) showing the spatial relationship between the second half (purple sticks) of the ED domain (⁹⁴⁷EDEDEEEEEEEDEDD⁹⁶¹) in the N1–T2 linker, the Kir6.1 positively charged residue cluster (blue spheres), and residues in SUR2B-NBD2 including K1348 of the Walker A motif (blue sphere), and Y1317 (blue sticks) and E1318 (purple sphere) in the A-loop, involved in Mg-nucleotide binding. The red box on the right shows dominant interactions observed during three independent 1-μs MD simulations in the absence of MgADP and which are disrupted in the presence of MgADP (red X) due to competition of the NBD2 residues for MgADP binding. The blue-circled + denotes Kir6.1 positively charged residue cluster. A and B are adapted from Fig. 5 A, and C from Fig. 6 B in Sung et al. (2021).