Structure of Na _V Ab/E96P. (A) Crystal structure of Na_VAb/E96P with the mutation shown in red. (B) Close-up view of Na_VAb/E96P structure in the activated state (teal). Residues E96P (red), Ile97, Leu98, Arg99 (R1), and Val100 (cyan) are shown as sticks with the σ_A-weighted 2F_O-F_C electron density map contoured at 1.0σ level overlaid (mesh). A DMPC lipid molecule interacting with E96P is shown as yellow sticks. (C) Comparison of Na_VAb/E96P structure (teal) with the structure of Na_VAb WT (light orange). The backbone carbonyl oxygen of Glu96 and the amide nitrogen of Val100 form a hydrogen bond (black dashes) that stabilizes the S4 helix in the WT structure. The E96P mutation breaks the helical conformation of S4 by inducing a longer distance between these atoms (red dashes). The side chain of E96P also interacts with the glycerol backbone (green) of DMPC. The van der Waals hemispheres are shown as dots. Na_VAb/I217C (PDB accession no. 3RVY) was used for the comparison as WT because the S3–S4 loop was not modeled in Na_VAbΔ28 (PDB accession no. 6MWA). (D) Homology model of Na_VAb/E96P structure in the resting state. The backbone carbonyl oxygen of E96P and the amide nitrogen of R1 form a hydrogen bond (black dashes) that stabilizes the 3₁₀ helix.