Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

(A) Representative micrograph from grid screening on a Falcon-3 detector (Talos-Arctica), showing detergent-solubilized GLIC particles. (B) Representative 2D class averages at 0.82 Å/px in a 256 × 256 pixel box and a 180 Å mask. (C) Overview of cryo-EM processing pipelines for data collected at pH 7 (blue), pH 5 (green), and pH 3 (lavender) (see the Materials and Methods section).

(A) Left: FSC curves for cryo-EM reconstructions at pH 7 before (red) and after (blue) applying a soft mask. Right: FSC curves for cross-validation between model and half-map 1 (light), model and half-map 2 (medium), and model and summed map (dark). (A, B) FSC curves represented as in panel (A) for the dataset at pH 5. (A, C) FSC curves represented as in panel (A) for the dataset at pH 3.

(A) Cryo-EM map for GLIC at pH 7 (left) and the distribution of orientation sampling after refinement, with low–high values represented by bar height and color (blue–red, right). (A, B) Map and distributions as in panel (A) for the dataset at pH 5. (A, C) Map and distributions as in panel (A) for the dataset at pH 3.

(A) Density (mesh) and corresponding atomic model (sticks, colored by heteroatom) for the M2 helix (E222–E243) at pH 7 (blue, left), pH 5 (green, center), and pH 3 (lavender, right). (A, B) Density and corresponding model, shown as in panel (A), for the β7 strand (P120–I128). Side chains that could not be definitively built at pH 7 (D122, Q124, and L126) are represented by Cβ atoms.

(A) Density colored by local resolution as in Fig 1B, for the dataset at pH 7. (A, B) Density as in panel (A), generated from a random subset of particles collected at pH 5, equal in number to the pH-7 dataset (86,000). (B, C) Density as in panel (B) from an equivalent subset of particles collected at pH 3.

Selected region of the crystal lattice of GLIC at low pH (PDB ID 4HFI), with three neighboring molecules (gray) packed in C121 symmetry. Zoom boxes at right show sliced overlays of state-1 reconstructions at pH 7 (blue), pH 5 (green), or pH 3 (lavender) with one crystallographic asymmetric unit (dark gray), revealing clashes of cryo-EM densities with the crystal-lattice neighbor (light gray). Crystal models and cryo-EM densities are shown as surfaces at 6 Å resolution.

(A) Overlay as in Fig 2A of previous X-ray structures crystallized under resting (white, PDB ID: 4NPQ) and activating (gray, PDB ID: 4HFI) conditions. Two adjacent subunits are viewed as ribbons from the channel pore, showing key motifs including the β1–β2 and Pro loops and M1–M4 helices from the principal subunit (P), and loop F from the complementary subunit (C). (A, B) Zoom views as in Fig 2C of the black-boxed region in panel (A), showing key side chains (sticks, colored by heteroatom) at a single domain interface in resting (white, left) and open (gray, right) X-ray structures. Dotted circle indicates the side chain of K33, which could not be definitively built in resting conditions. Center panel shows major backbone transitions from overlaid resting to open states (orange arrows).

(A) Root mean-squared deviations over time for Cα-atoms of the ECD (solid) and TMD (dotted) in four replicate 1-μs molecular dynamics simulations of cryo-EM structures determined at pH 7 (blue), pH 5 (green), and pH 3 (lavender). Simulations were performed with side chain charges approximating resting (deprotonated, top) or activating (protonated, bottom) conditions (14). Reference simulations of resting (gray, top) and open (black, bottom) X-ray structures are shown at right. (A, B) Hydration at the hydrophobic gate during simulations under deprotonated (solid) or protonated (striped) conditions as depicted in panel (A), quantified by water occupancy between I233 (I9′) and A237 (A13′) in the channel pore. (B, C) Loop-F root mean-squared deviation during simulations as in panel (B), showing diminished deviations in pH-3 cryo-EM models, and in low- versus neutral-pH X-ray models. (B, C) In panels (B, C), histograms represent median ± 95% confidence interval over all simulations in the corresponding condition.

(A) Views as in Fig 1B of pH-3 state-1 (lavender) and state-2 (purple) cryo-EM densities, shown from the membrane plane (left) or extracellular side (right). Right-hand views are shown for the ECD (above), with purple arrows indicating inward contraction and counter-clockwise untwisting of the ECD in state 2 relative to state 1; and for the TMD (below), with white arrows indicating outward tilting of the upper M2 helices. (B) Histograms indicating parallel trends in ECD contraction (left) and untwisting (right) from resting (gray) to open (black) X-ray structures, and from pH-3 state-1 (lavender) to state-2 (purple) cryo-EM structures. Superimposed error bars show median values ± 25% confidence intervals from molecular dynamics simulations of the three fully built models.

(A) Overview of cryo-EM state 2 processing pipeline for data collected at pH 7 (dark blue). The 3D classification with 50 classes was carried out on the largest subset of fairly clean particles (see the Materials and Methods section). (A, B) Same as in (A) but for a dataset at pH 5 (dark green). (A, C) Same as in (A) but for a dataset at pH 3 (dark purple).

(A) Overlaid side view of backbone ribbons representing two adjacent subunits from GLIC cryo-EM models at pH 7 (light and dark blue), pH 5 (light and dark green), and pH 7 (light gray, PDB ID: 4NPQ) and pH 4 (dark gray, PDB ID: 4HFI). (B) Comparisons of the electrostatic network between the X-ray structures and pH-7 state 1 (blue), state 2 (dark blue), and pH-5 state 1 (green) and state 2 (dark green). The relevant secondary structure elements are labeled. Density of each structure is shown in mesh in their respective color. (C) Pore profiles of cryo-EM static structures (state 1 and state 2) for pH 7 (blue), pH 5 (green) and pH 3 (purple) as well as resting (gray) and open (black) X-ray structures. (D, E, F, G, H) Pore profile of the molecular dynamics derived structures compared to a static structure for resting X-ray, open X-ray Pore profiles of structures (state 1 and state 2) for pH 7 (blue), pH 5 (green), and pH 3 (purple) compared to the molecular dynamics derived structures under activating and resting conditions.