Single particle cryo-EM of ELIC in liposomes.

(A) Schematic of the fluorescence stopped-flow liposomal flux assay: a sequential mixing experiment is performed. ELIC proteoliposomes are mixed with agonist followed by thallium (Tl+) after a variable delay time. ELIC activity at each delay time is measured as the rate of fluorescence quenching by the influx of Tl+. Shown is WT ELIC and ELIC5 activity after mixing with 10 mM propylamine over varying delay times (n = 3-6, ± SD). (B) Representative cryo-EM micrograph of WT ELIC in 2:1:1 POPC:POPE:POPG liposomes and 2D class averages. (C) Cryo-EM maps of WT ELIC without agonist in liposomes showing inward-and outward-facing orientations. The lipid bilayer density is shown at a lower contour level for illustration.

Structure and pore conformation of resting, activated, and desensitized states.

(A) Cryo-EM maps of WT ELIC without agonist (resting state), ELIC5 with 10 mM propylamine (activated state), and WT ELIC with 10 mM propylamine (desensitized state) in liposomes. In the ELIC5 + Agonist map, red shows non-protein densities. (B) Two opposing subunits from models of the structures shown in (A) along with a dot plot of the pore dimensions generated using HOLE [34]. Shown are the side chains for F247 (16’), L240 (9’) and Q233 (2’).

Single particle cryo-EM of ELIC in spNW25 nanodiscs.

(A) Cryo-EM micrograph of WT ELIC with 10 mM propylamine in spNW25 nanodiscs. The circles indicate top-down views of spNW25 nanodiscs with multiple ELIC ion channels at the rim of the nanodisc. (B) 2D class averages of WT ELIC in spNW25. (C) C1 cryo-EM map of agonist-bound WT ELIC in spNW25 at low contour showing the nanodisc scaffold density (gray). M4 is resolved in one of the ELIC subunits colored yellow. (D) Displays of the local resolution for the map in (C). Shown are the aspects of ELIC facing the lipid bilayer and the nanodisc scaffold. (E) C1 cryo-EM map of agonist-bound WT ELIC in spMSP1D1 at low contour showing the nanodisc scaffold density (gray). M4 densities can be appreciated in all subunits except one of the lipid bilayer-facing subunits (star). (F) Displays of the local resolution for the map in (E). Shown are the aspects of ELIC facing the lipid bilayer and the nanodisc scaffold.

Comparison of ELIC structures in liposomes and nanodiscs.

(A) Comparison of the M1, M2, and M3 helices in structures of WT ELIC with agonist in liposomes, spMSP1D1 and spNW25. Shown in M2 is the side chain of L240 and the approximate location of the ion conduction pore relative to M2. Images are from a global superposition of the structures. (B) Two opposing subunits from structures of WT ELIC with agonist in liposomes and spNW25. Shown are dot plots of the pore dimensions generated using HOLE [34], and the side chains for F247 (16’), L240 (9’) and Q233 (2’). (C) Comparison of M4 in WT ELIC in liposomes, WT ELIC with agonist in liposomes, and WT ELIC with agonist in spMSP1D1 showing a side view and top view (from extracellular side) of the helix. The approximate location of the lipid bilayer and ion conduction pore are indicated relative to M4. Images are from a global superposition of the structures. (D) Same representation as (C) comparing M4 in WT ELIC with agonist in liposomes, ELIC5 with agonist in liposomes, and ELIC5 with agonist in MSP1E3D1.

Summary of single particle cryo-EM analysis of WT ELIC in liposomes.

After multiple rounds of heterogeneous refinement in CryoSPARC to separate particles into inward-and outward-facing populations, multiple rounds of 3D classification were performed in Relion-5 to yield the final maps. The results from the final heterogeneous refinement and 3D classification jobs are shown.

Summary of single particle cryo-EM analysis of ELIC5 with agonist in liposomes, similar to Supplementary Figure 1.

Summary of single particle cryo-EM analysis of WT ELIC with agonist in liposomes, similar to Supplementary Figure 1.

Display of local resolution on sharpened maps and FSC curves from outward-facing and inward-facing ELIC structures in liposomes.

The final map of outward-facing ELIC5 with agonist in liposomes was obtained from CryoSPARC. The remaining maps were obtained from Relion-5.

Comparison of resting (WT apo), activated (ELIC5 + agonist) and desensitized (WT + agonist) structures of ELIC in liposomes.

All images are from a global superposition of the structures. (A) View of the agonist/orthosteric binding site with bound propylamine. Loop C and three aromatic residues that interact with propylamine are labeled. The activated structure shows a contraction of the agonist binding site compared to the resting structure. (B) View of the extracellular domain (ECD) from the extracellular side along the pore axis. The activated structure shows a counter-clockwise twisting and slight contraction of the ECD. (C) View of the ECD-TMD interface. The β1-β2, β6-β7, and β8-β9 loops are labeled along with M4, and M3 and M1 from adjacent subunits. The ECD interfacial loops are similar between the activated and desensitized structures, but the transmembrane helices show a tilting and translation away from the pore axis that is greater in the activated compared to the desensitized structure. (D) View of the TMD from the extracellular side along the pore axis. (E) View of the pore-lining M2 showing the rotation of the F247 and L240 in the activated structure compared to the resting and desensitized structures.

View of the cryo-EM map of WT ELIC with agonist in liposomes showing the fit for State 1 and State 2 models, which differ primarily in the side chain orientation of F247 and Y248.

In State 1, F247 orients towards the pore axis, while in State 2, F247 orients towards the adjacent subunit.

Cryo-EM map of ELIC5 with agonist in liposomes with non-protein lipid-like densities colored in red.

The lipid-like densities in the green box are shown on the right along with the phospholipid from PDB 8VUW (structure of agonist-bound ELIC5 in MSP1E3D1 nanodiscs). This image was produced by fitting ELIC5 from 8VUW in the ELIC5 map from liposomes, and displaying the phospholipid from 8VUW with the non-protein densities from the map of ELIC5 in liposomes.

Summary of single particle cryo-EM analysis of WT ELIC with agonist in spNW25 nanodiscs.

2D class averages were separated into single pentamers or dimers of pentamers. The particles consisting of single pentamers were processed with C1 symmetry in CryoSPARC only, or with C5 symmetry in CryoSPARC and Relion-5. Shown are the FSC curves for both C1 and C5 reconstructions, and the local resolution of the C5 map. The C1 map did not show significant asymmetry in the protein structure except for differences in the local resolution. Therefore, a model was only produced based on the C5 reconstruction, which had better resolution. The dimers of pentamers were separately processed in CryoSPARC with C2 symmetry, and this did not yield a high-resolution reconstruction.

Summary of cryo-EM data collection and refinement parameters.

Distance along the pore axis (i.e. perpendicular to the lipid membrane) between the indicated residues (Cα atoms) for each structure.

The measurements show changes in the height of each transmembrane helix along the pore axis.