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	Figure 1. Structurally ordered water molecules in the pore of GLIC. (A) Enlarged representation of the pore with the M2 helices shown as a cartoon and the side chains of the pore-lining residues shown as sticks. A well resolved self-stabilized ‘tight pentagon' of water molecules is present at the level of Ser6′ (left top). A Na+ atom and five bound water molecules are observed at the intracellular end of the pore (left bottom). Detergents are shown as sticks, waters and Na+ atoms as spheres. The blue mesh is the maximum likelihood 2mFo–DFc map contoured at a level of 1.5σ. For clarity, the electron density surrounding the Na+ has been removed from the left bottom panel. (B) Interaction network between the water molecules, the Na+ and the pore-lining M2 residues. The alternative conformation that is adopted by Glu-2′ is also represented (yellow).
	Figure 2. Residual electron density in the pore for a set of deposited GLIC structures at different resolutions (A, C, D, E and F) and for GluCl (B). The difference Fourrier (Fo–Fc) omit map (contoured at 3σ) as calculated from a model where detergents, Na+ and water molecules were not included during refinement. As the resolution decreases, the density around the water molecules no longer appears like individual spheres but rather like a fused blob. While the tight pentagon is systematically observed in the electron density, the second layer of water molecules located on top of it is unequally distributed among these structures, especially in the GLIC 3UUB entry, where a complete water pentagon can be built on top of the tight pentagon (blue). The Na+ ion is systematically observed in the residual electron density but with a corresponding peak of variable intensity and slightly different position. These differences might be explained by slight variations of the conditions used during crystal freezing. Superimposing the M2-helices in the GLIC (grey) and GluCl (pink) structures reveal a very strong structure conservation in this region of the pore (B). When the GluCl structure was solved, the authors observed residual electron density in the Fo–Fc map that was built tentatively as a chloride ion. We hypothesize that this residual density might as well be explained by several water molecules, presumably ordered in a similar pentagonal fashion as we observed in GLIC. The blue mesh is the Fo–Fc map (contoured at 2.5σ) obtained when the chloride ion built in the structure of GluCl was omitted during refinement. Iodide ions observed in GluCl (3RIA) are shown as purple circles.
	Figure 3. Cation, anion and detergent binding sites in the pore of GLIC. Central view: Cartoon representation of the GLIC structure viewed from the side with the solvent-accessible surface of the channel shown as a mesh (yellow). The top and bottom panels represent enlarged views of the channel vestibule and of the transmembrane pore showing the interactions between the protein and the solvent molecules. Acetate (orange) and sulphate (grey) molecules are shown as ball and sticks. (Middle-right panel) The green mesh is the Fo–Fc difference map (contoured at 3σ) calculated when the sulphates were omitted during refinement. (Bottom-left panel) Orientation of the dodecyl-β-D-seleno-maltoside (Se-DDM). The green mesh is the Fo–Fc difference map (contoured at 3σ) calculated when detergents were omitted during refinement and the blue mesh is the anomalous map for selenium contoured at 4σ. (Bottom right) Ion binding sites at the selectivity filter. The anomalous maps calculated for Br− (red), Cs+ (blue) or Rb+ (cyan) are superimposed and shown as a mesh (contoured at a level of 4σ) with the corresponding ions shown as spheres.
	Figure 4. Electrophysiology of Ser6′ mutants. (A) Voltage-clamp current traces recorded from GLIC wt and the S6′G, S6′T, S6′V and S6′T/I9′L variants expressed in Xenopus oocytes, showing the effect of switching extracellular pH from 8 to 5. (B) Immunofluorescence microscopy data showing that all GLIC variants express at the oocyte surface. (C) SDS denaturating gel experiment in reducing conditions showing that all GLIC variants express in E. coli. (D) Traces of currents recorded from an outside-out patch from BHK cells transfected with a mixture of cDNAs coding for GLIC S6′G and GLIC wt, in a 4 to 1 S6′G to wt DNA ratio (a=0.8). Currents recorded at extracellular pH 8 (upper trace), then pH 5, then pH 5 with 0.1 mM picrotoxinin, pH 5 after toxin wash-out, and pH 8, on the same patch. Holding potential −100 mV. The patch input resistance was 0.6 tΩ (closed level at pH 5). The current levels indicated right to the traces correspond to all channels closed (c), and to one channel open (o) for the prominent, large conductance events measured in F (4 pS), arising from S/G6′-heteropentamers. (E) Trace of current recorded from an outside-out patch from a cell expressing wt GLIC channels. Holding potential of −80 mV. The lower part of the recording has been skipped out from 1.4 pA under the closed channel current level, in order to emphasize the one open channel level for wt GLIC. (F) Plots of single-channel current versus holding potential values, for wt GLIC (grey circles) and for GLIC S/G6′-heteropentamers (black circles) corresponding to the open level emphasized in (D). Number of patches and error bars indicating standard deviations are shown for each voltage. Data were accumulated from four patches (wt), and from eight patches from cells with a=0.8 or a=0.94.
	Figure 5. Ion atmosphere and water binding sites predicted by full-atom MD. Enlarged representation of the pore showing the averaged densities calculated over 200 ns for water molecules (green mesh and yellow surface contoured at increasing density starting from the bulk density shown as white transparent surface), Na+ (blue mesh) and Cl− (red mesh). For comparison, experimentally observed water molecules, anions and cations are shown as cpk spheres (coloured in black, red and blue, respectively). (Right bottom panel) Comparing the hydration profile observed when the side chain of Ser6′ adopts an α or a β conformation (see Supplementary Information for details). (Right top panel) Detailed view of the network of water molecules that covers the edges of the hydrophobic half of the channel (distances are measured in Å). Primary and secondary layers of water molecules are shown as red and pink spheres, respectively.
	Figure 6. Hydration profiles of GLIC′s pore in WT and Ser6′ mutant simulations. Hydration traces of the pore of GLIC during the 20-ns equilibration (with restraints on the Cα positions) and the following 100 ns production. The origin of the z-axis (0 Å) corresponds to the position of Ile9′. The hydration level is represented using a colour scale from dark blue (low hydration) to cyan, green and yellow (strong hydration). The absence of water is depicted by a white colour. On the right panel, snapshots corresponding to the last frame of the production are shown for the pore of GLIC wt and S6′T mutant. Water molecules are shown as spheres.
	Figure 7. Hydration profile of Na+ crossing the TMD. (A) First hydration shell composition (calculated with a distance cutoff of 3.2 Å for all Na+ ligands and averaged over a time window of 0.25 ns). The colour code is the following: water (cyan), Cl− (red), Na+ (blue) and oxygen atoms from the side chains of Ser6′ (green), Thr2′ (yellow) and Glu-2′ (orange). (B) Z-dependent hydration profile of the pulled Na+. In each slice of 1 Å, the water density of the ion's first water shell was computed along the z-axis. Selected pore structures (from a to f) are represented on the bottom panel (C) for six different snapshots; the pulled ion is displayed as a yellow sphere, water molecules are shown as red and white sticks, and water molecules present in the first hydration shell are represented by larger sticks. Pore-lining residues are shown in cyan. Transparent spheres represent waters and ions present in the crystal structure: small red spheres for water and large spheres for ions (red for Br− and blue for Cs+ and Rb+).
	Figure 8. Optimized set of configurations of the Ser6′ and Thr2′ side chains. Five representative configurations out of the 72 generated ones of GLIC (A) and GluCl (B) are shown. They maximize the permeant ion (cation for GLIC, anion for GluCl) density at five altitudes in the pore (indicated by a white sphere on the pore axis), from its extracellular to its intracellular end (left to right). The hydroxyl groups of residues 2′ and 6′ are shown as spheres. The favourable region for the permeant ion is shown as a mesh isocontoured at the bulk density value (200 mM) (blue for cation, red for anion). The regions where the permeant ion density is higher than 20 times the bulk value are shown as a blue surface for cation, red surface for anion. The altitudes of experimentally observed ions in GLIC are indicated by dashed lines. On the left of the figure, vertical bars indicate the regions where the permeant ion density is affected by either residue 2′ or 6′.

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