Fig. 4. BamA β-barrel dynamics are regulated by the membrane composition and POTRA. (a) Results of all-atom MD simulations of full-length BamA and POTRA⁻ BamA in PC:PE and PC:PG membranes. Atomic displacement (B-factor analysis) is rendered as a sausage representation (red, highest RMSD; dark blue, lowest RMSD). In PC:PE (i), POTRA, L4, and L6 exhibit high structural dynamicity, which allosterically translates to increased dynamicity of N-terminal β3–β6. POTRA deletion abolishes this plasticity in the TMD (ii), limiting the conformational flexibility to L4 and L6. In contrast, PC:PG membranes restrict the conformational dynamicity to only POTRA1 in full-length BamA (iii) and additionally L6 (iii) and (iv). PE increases BamA TMD and POTRA plasticity, while the TMD is conformationally rigid in PG, irrespective of the POTRA. (b, top panels) Free energy landscape (FEL) of full-length BamA (residues 21–810) shows a marked difference in the presence of PC:PE (top left) and PC:PG (top right). Full-length BamA displays greater conformational freedom in PC:PE (higher RMSD; broader FEL profile) than in PC:PG. A narrower FEL profile suggests that PC:PG restricts the β-barrel to concurrently sample fewer conformational ensembles. (b, bottom panels) Removal of the POTRA impacts conformational accessibility of the TMD (BamA^424–810). The TMD is rigid both in PC:PE and PC:PG (narrower FEL). (c) Comparison of per-residue root-mean-square fluctuation (RMSF) in PC:PE (teal) and PC:PG (purple). Comparison of full-length BamA RMSF in both membranes (top) shows considerable difference in the POTRA region. This is in excellent agreement with the structures in (i) and (iii). β-Strand regions are indicated as gray shading. Comparison of RMSF of full-length and POTRA⁻ BamA in PC:PE (middle) and PC:PG (bottom) shows that the removal of POTRA in BamA^424–810 decreases fluctuations of the TMD and abolishes fluctuations at loop 5 (residues T⁶⁰⁰–K⁶¹⁰; details in Fig. S14) only in PC:PE (middle). PC:PG (bottom) exerts only a marginal influence on the RMSF, irrespective of whether POTRA is retained. Overall, simulations indicate that PE increases plasticity of the β-barrel domain, and POTRA dynamicity.