The outside of the membrane-inserted part of the β-barrel is almost entirely hydrophobic (Figure 5A,E) and covered by the toroid density of disordered amphipol that replaces the membrane lipid (Figure 1B), as in the cryoEM structures of amphipol-solubilized membrane proteins (Althoff et al., 2011; Liao et al., 2013). The inside pore surface is highly polar, with three aspartates and two glutamates forming a 15 by 9 Å patch of negative charge, flanked above and below by positive charges (Figure 5B,F,G). Apart from the hydrogen bonds between the 168 β-strands in the barrel and the interactions of helix α3a with D1 and β-hairpin one (Figure 2E, Figure 2—figure supplement 4), three other factors contribute to pore stability: (1) the surface and charge complementarity of the membrane-inserted monomers (Figure 5C); (2) the alternating positive and negative charges of helices α13a and α13b in the α-barrel (Figure 5D); and (3) ionic interactions between charged sidechains in adjacent β-strands of the pore barrel (Figure 5G). In particular, Asp168 and Glu170 in β-strand β7 are in a good position for forming a salt bridge with Lys271 in β-strand β10 of the next-door monomer (Figure 5G).10.7554/eLife.23644.015Figure 5.Charge distribution in the PLY pore complex.(A) Positive (blue) and negative charges (red) are evenly distributed on the polar outer surface of the pore complex. The membrane-inserted region is marked by a band of neutral hydrophobic residues (arrows). (B) The inner surface of the pore complex is highly charged. (C) Contact surfaces of adjacent PLY monomers in the pore complex. (D) Charge complementarity of the two helices in the helix-turn-helix motif forming the internal α-barrel. Positive charges are shown in blue and negative charges in red. (E) The membrane-inserted region of the β-barrel is hydrophobic on the outside and negatively charged on the inside (F). The inset (G) shows map density for the salt bridge between Asp168 of one PLY monomer with Lys271 in the next monomer along the ring. Glutamates and aspartates forming the negatively charged patches on the inner surface of the β-barrel are drawn as sticks. The map is contoured at 5.0 and 6.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.015