In the outward-facing Glt_Ph and EAAT1 in complex with blockers TBOA and TFB-TBOA or Na⁺ ions only, HP2 opens to various degrees, enabling access to the substrate-binding site (Canul-Tec et al., 2017; Boudker et al., 2007; Verdon et al., 2014). To picture gating in the IFS, we imaged the Glt_Ph reconstituted into MSP1E3 nanodiscs in the presence of various ligands by single-particle Cryo-EM. Because wild type Glt_Ph strongly prefers the OFS in detergent and lipid environments (Akyuz et al., 2015; Huang et al., 2020; Ruan et al., 2017; Georgieva et al., 2013; Hänelt et al., 2013), we used a variant of Glt_Ph, conformationally constrained in the IFS by crosslinking of cysteine residues placed into the transport and scaffold domains, Glt_Ph-K55C/A364C (Glt_Ph^IFS) (Reyes et al., 2009). Earlier crystal structures of Glt_Ph^IFS pictured the position of the transport domain that was very similar to those visualized in unconstrained inward-facing Glt_Ph mutants (Akyuz et al., 2015; Verdon and Boudker, 2012).

We determined the structures of Glt_Ph^IFS free of ligands (Glt_Ph ^IFS-Apo-open) or in complex with Na⁺ ions (Glt_Ph^IFS-Na) and bound to L-asp (Glt_Ph^IFS-Asp), TBOA (Glt_Ph^IFS-TBOA), TFB-TBOA (Glt_Ph^IFS-TFB-TBOA), and the wild type outward-facing Glt_Ph in complex with TBOA (Glt_Ph^OFS-TBOA) to 3.52, 3.66, 3.05, 3.39, 3.71, and 3.66 Å resolution, respectively (Materials and methods, Figure 1—figure supplements 2–4, and Table 1). The Cryo-EM Glt_Ph^IFS-Asp structure was nearly identical to the earlier crystal structure (RMSD of 1.0 Å) (Reyes et al., 2009). The transport domain was well packed against the scaffold primarily through interactions of HP2 and the extracellular part of TM8 (TM8a) with the scaffold TMs 2, 4, and 5. The central axis of the roughly cylindrical transport domain formed a ~ 35 ° angle with the membrane normal (Figure 1a,b). HP2 was closed over the substrate-binding site and packing between the transport and scaffold domains left no space for it to open. A similar inter-domain orientation and packing were also observed in a previously solved crystal structure of the occluded apo Glt_Ph^IFS (Glt_Ph^IFS-Apo-closed, PDB code 4P19, Figure 1a; Verdon et al., 2014). In the new Cryo-EM structures of Glt_Ph^IFS-Na, Glt_Ph^IFS-Apo-open, Glt_Ph^IFS-TBOA, and Glt_Ph^IFS-TFB-TBOA, approximately the same regions of HP2 and TM8a remained juxtaposed against the scaffold. However, the bulk of the transport domain swung out away from HP2 and the scaffold to different extents (Figure 1a) with the largest angle between the transport domain and the membrane normal of ~ 47° in Glt_Ph^IFS-TFB-TBOA (Figure 1b). Together, the crystal and Cryo-EM structures define gating mechanisms in Glt_Ph on the extracellular and cytoplasmic sides (Figure 1c, Video 1). In the OFS, the bulk of the transport domain remains mostly static relative to the scaffold, and the labile HP2 serves as the extracellular gate. In the IFS, HP2 can maintain interactions with the scaffold, while the bulk of the transport domain swings away to allow access to the binding site. Notably, in a crystal structure of a gain-of-function aspartate-bound mutant Glt_Ph^IFS-R276S/M395R, the transport domain is positioned at ~ 45 ° angle (Akyuz et al., 2015), similar to the Glt_Ph ^IFS-TFB-TBOA Cryo-EM structure. However, in Glt_Ph^IFS-R276S/M395R, HP2 remains closed over the binding site and a large lipid-filled gap forms between the transport and scaffold domains. It is currently unclear whether the transport domain first swings away from the scaffold providing space for the consequent HP2 opening or whether HP2 remains in place while the bulk of the domain swings out in a ‘wag-the-dog’ manner.

TBOA and TFB-TBOA blockers share the amino acid backbone with L-asp but are decorated on β-carbon with one and two benzyl rings, respectively, that cannot fit within the confines of the substrate-binding site. They block transport by binding to the outward-facing Glt_Ph, Glt_Tk, or EAATs and arresting HP2 in an open conformation (Boudker et al., 2007; Canul-Tec et al., 2017; Arkhipova et al., 2020). Our Cryo-EM structure of the outward-facing Glt_Ph^OFS-TBOA in nanodisc confirmed that the transporter took the same conformation in the absence of crystal contacts in a lipid bilayer (RMSD = 1.0 Å, PDB accession code 2NWW) (Boudker et al., 2007; Figure 2—figure supplement 1a). TBOA and the related L-β-threo-benzyl-aspartate (TBA) bind to the IFS of Glt_Ph (Reyes et al., 2013; Oh and Boudker, 2018). We used isothermal titration calorimetry to show that TFB-TBOA and TBOA bind to Glt_Ph^IFS in 200 mM NaCl with 1:1 stoichiometry and the dissociation constants (K_Ds) of 3.8 and 6.5 μM, respectively (Figure 2—figure supplement 2a, b). We then determined the structures of the Glt_Ph^IFS complexes with the blockers TFB-TBOA and TBOA under saturating conditions in the presence of 10 mM inhibitors.

In the Glt_Ph^IFS-TFB-TBOA structure, TFB-TBOA density was well resolved, and we modeled the inhibitor in its binding site (Figure 2a). We also modeled L-asp into the excess density in the binding site of Glt_Ph^IFS-Asp (Figure 2—figure supplement 1b). The bound L-asp and TFB-TBOA share some critical interactions (Figure 2c). Thus, R397 coordinates the side chain carboxylates of aspartate moieties, and D394 coordinates the amino groups. However, TFB-TBOA assumes a different rotomer, leading to a displacement of the backbone carboxylate and the loss of coordination by the highly conserved N401. The aromatic rings of TFB-TBOA protrude from the ligand-binding site and lodge in between the transport and scaffold domains (Figure 2a, Figure 2—figure supplement 1d). Most strikingly, HP2 takes a wide-open conformation that is essentially the same as in the outward-facing Glt_Ph-TBOA complex (Figure 2—figure supplement 1c). Interestingly, HP2 was in the same conformation also in an R397C Glt_Ph mutant bound to glutamine or benzyl-cysteine. In these structures, the ligands made virtually no interactions with the hairpin but introduced steric clashes disallowing closure (Scopelliti et al., 2018). Therefore, it appears that the hairpin intrinsically favors this open conformation.

Surprisingly, HP2 does not open in the same way in Glt_Ph^IFS-TBOA. Instead, the hairpin remains mostly closed, but its N-terminal arm separates from the C-terminal arm. The bound TBOA assumes a similar rotomer as TFB-TBOA, though N401 still coordinates the backbone carboxylate (Figure 2c). The C-terminal arm coordinates the sidechain carboxylate of the aspartate moiety as in Glt_Ph^IFS-Asp. The TBOA benzyl group inserts in between the two arms packing against M311 and M362 sidechains (Figure 2b,c). The N-terminal arm movement disrupts the Na2 binding site, consistent with previous observations that binding of TBOA and TBA to the IFS of the transporter required only two Na⁺ ions (Reyes et al., 2013; Oh and Boudker, 2018). The movement creates a small opening into the cytoplasmic milieu between the tips of HP1 and HP2. It is not clear whether this conformation reflects a functional state. Perhaps, it recapitulates a transient transporter state, in which a Na⁺ ion has already left the Na2 site while the substrate and two other Na⁺ ions are still bound. Water might use the cytoplasmic opening to reach and eventually displace the remaining solutes.

These structures collectively show that in Glt_Ph^IFS, bulky competitive blockers can be accommodated either by opening HP2 or by parting its N- and C-terminal arms (Figure 2d). Since the OFS and IFS share the same binding pocket for the substrate and competitive inhibitors, it is likely that the new inhibitor binding mode with parted HP2 arms can be sampled in the OFS as well. This mode of blocker binding might provide new pharmacological avenues for the inhibition of human glutamate transporters.

To further explore the gating mechanism, we aimed to resolve a structure of Na⁺ only-bound Glt_Ph^IFS and imaged nanodisc-reconstituted Glt_Ph^IFS frozen in the presence of 200 mM NaCl (Figure 1—figure supplement 3b and 4, and Table 1). We isolated two distinct structural classes of Glt_Ph^IFS protomers after symmetry expansion and classification without alignment. The structural heterogeneity was not surprising in retrospect because Na⁺ concentration in the sample was close to the dissociation constant measured for Glt_Ph^IFS (Reyes et al., 2013). Thus, we observed both Na⁺-bound (Glt_Ph^IFS-Na) and apo (Glt_Ph^IFS-Apo-open) states. We assigned these states based on the conformations of the conserved non-helical NMD motif (residues 310–312) in TM7, which coordinates Na⁺ ions in the Na1 and Na3 sites, and TM3, part of the Na3 site (Figure 3—figure supplement 1a; Boudker et al., 2007; Guskov et al., 2016). In particular, the M311 sidechain protrudes toward the L-asp and Na2 sites in Glt_Ph^IFS-Na and Glt_Ph^IFS-Asp structures. In contrast, it flips out toward TM3 in our Glt_Ph^IFS-Apo-open structure and the previous Glt_Ph^IFS-Apo-closed crystal structure (Verdon et al., 2014). We did not observe density for Na⁺ ions in the Na1 and Na3 sites of Glt_Ph^IFS-Na. However, all ion-coordinating residues are positioned similarly to Glt_Ph^IFS-Asp (Figure 3—figure supplement 1b). Notably, Na1 is coordinated in Glt_Ph^IFS-Asp, in part, by an occluded water molecule (Figure 3—figure supplement 1b). In Glt_Ph^IFS-Na, the water is no longer occluded and is part of an aqueous cavity (Figure 3a). We conclude that ions likely occupy Na1 and Na3 sites, but the Na1 site might be in rapid equilibrium with the solution.

The Cryo-EM Glt_Ph^IFS-Apo-open structure differs significantly from the occluded Glt_Ph ^IFS-Apo-closed crystal structure in that the substrate-binding site is open and hydrated. The opening resembles that in Glt_Ph^IFS-Na compared to the occluded Glt_Ph^IFS-Asp (Figure 3a,b) and shares the overall mechanism: HP2 remains in contact with the scaffold while the rest of the transport domain swings out (Figure 1c). From the transport domain viewpoint, the conformational changes lead to a similar HP2 opening (Figure 3c,d, Figure 3—figure supplement 2a). Interestingly, in Glt_Ph^IFS-Na, there is also a small shift of HP1 away from the substrate-binding site, possibly increasing water access to Na1. A similar small movement of the otherwise rigid HP1 was observed in the crystals of apo Glt_Ph^IFS grown in an alkali-free buffer (Verdon et al., 2014).

Two residues in the transport domain - M311 and R397 - move significantly during gating and might couple solute binding and release to large-scale conformational changes. Here we consider a sequence of structural events, which might underlie ion and substrate release in the IFS (Figure 1c), starting with Glt_Ph^IFS-Asp and going to Glt_Ph^IFS-Na, Glt_Ph^IFS-Apo-open, and Glt_Ph^IFS-Apo-closed (Video 2). In Glt_Ph^IFS-Asp, the R397 side chain extends upward, toward the extracellular side of the membrane, allowing D390 to coordinate its guanidinium group. Thus positioned, R397 makes space for L-asp and coordinates its sidechain carboxylate, while D394 coordinates its amino group (Figure 3e). M311 protrudes into the binding site and coordinates Na2 (Figure 3—figure supplement 2). Extensive interaction of HP2 with the bound L-asp and Na2 favor the closed conformation (Figure 2c). HP2 opening accompanies L-asp and Na2 release (Glt_Ph^IFS-Na). R397 is now clamped between D390 and D394, while M311 remains in place (Figure 3e, Figure 3—figure supplement 2b). The consequent release of Na1 and Na3 leads to a restructuring of the NMD motif and outward rotation of M311, which now packs against the open HP2 of Glt_Ph^IFS-Apo-open (Figure 3—figure supplement 2b). The guanidinium group of R397 remains between D390 and D394. To achieve the closed apo state, M311 swings further out into the lipid bilayer, allowing HP2 to close. R397 descends deep into the binding pocket, coordinated now only by D394, and is poised to make direct or through-water interactions with carbonyl oxygens of the closed tip of HP2. Steric hindrance of M311 and more positive local electrostatics may prevent R397 from entering the aspartate-binding site and closing HP2 in Na⁺-only-bound Glt_Ph^IFS. Physiologically, such Na⁺-bound occluded states should be avoided to prevent Na⁺ leaks.

Interestingly, in our Cryo-EM analysis, we did not find any Glt_Ph^IFS-Apo-closed structures previously visualized by crystallography. It might be that the open conformation of the apo Glt_Ph^IFS is the preferred state of the transporter and that the Glt_Ph^IFS-Apo-closed state is assumed only transiently, before the outward transition of the transport domain. Packing crystal contacts, which include extensive interactions between the cytoplasmic sides of the transport domains (Verdon et al., 2014), might have stabilized the closed conformation.

HP2 and TM8a comprise most of the transport domain surface interacting with the scaffold in Glt_Ph inward-facing states. Strikingly, in each of our IFS structures, HP2 takes a different conformation (Figure 4—figure supplement 1a). These are similar in structures with occupied Na1 and Na3 sites, that is in complexes with Na⁺ ions only and with L-asp, TBOA, or TFB-TBOA. The differences are mostly around the tip of HP2 near the L-asp and Na2 sites (Figure 4—figure supplement 1a and b). In contrast, the helices restructure significantly in the apo conformations, particularly in Glt_Ph^IFS-Apo-open (Figure 4—figure supplement 1a and c). When we superimposed all IFS structures, aligning them on the scaffold domain, we observed that the HP2/TM8a motifs present the same bulky hydrophobic residues flanking the flexible tips for interactions with the scaffold: L347, I361, and L378 form virtually the same spatial arrangement. Only in Glt_Ph^IFS-Apo-open, I350 replaces L347 because the HP2/TM8a motif, particularly the HP2 N-terminal arm, moves outward (Figure 4—figure supplement 1d and e).

Thus, the positions of the HP2 tip on the domain interface are mostly conserved. The structural differences in the hairpins then lead to their different orientations relative to the scaffold and different positions of the transport domains, which lean away and rotate to different extents (Figure 1a,b). The rotation is small for Glt_Ph^IFS-Asp, relative to Glt_Ph^IFS-Apo-closed (7°), but is significant for Glt_Ph^IFS-Na (23°), and Glt_Ph^IFS-TFB-TBOA (29°) (Figure 1a,b). A consequence of these differences is that the bulky residues in the HP2 N-terminal arm, L339, L343, L347, and I350 make more extensive interactions with the scaffold TMs 4a and 4 c in Glt_Ph^IFS-Na and Glt_Ph^IFS-TFB-TBOA compared to other structures. Furthermore, interaction areas between HP2/TM8 and the scaffold domain differ, with Glt_Ph^IFS-Apo-closed and Glt_Ph^IFS-Asp structures having the smallest areas of 1086 and 1076 Ų, respectively, and Glt_Ph^IFS-Na showing the largest increase of ~ 400 Ų (Figure 4).

The interdomain interface disruption is a prerequisite for the transport domain translocation from the inward- to the outward-facing position. Therefore, altered geometry of the interface and larger interaction area may explain why translocation is inhibited by blockers TBOA and TFB-TBOA, or in the transport domain bound to Na⁺ ions only. While it is not possible to translate interaction areas into energies, it is notable that translocation-competent closed apo and L-asp-bound states show the smallest areas. Consistently, the crystal structure of the gain-of-function mutant R276S/M395R in the IFS (Akyuz et al., 2015) shows a domain interface area of 543 Ų, about half of the Glt_Ph^IFS-Asp, and a translocation rate several-fold faster than the wild type transporter.

The Cryo-EM structures of the outward- and inward-facing states of Glt_Ph are overall similar to the crystal structures. However, they differ in the N-terminus, which is unstructured in crystals but forms a short amphipathic helix positioned on the surface of the nanodiscs in the Cryo-EM OFS and IFS structures (Figure 1—figure supplement 1). A similar helix was also observed in crystallized EAAT1 (Canul-Tec et al., 2017). We find highly ordered lipid molecules between the N-terminal helix and the rest of the scaffold at positions conserved in all structures (Lipid_In, Figure 5a and Figure 5—figure supplement 1). It seems likely that the helix anchors the scaffold domain in the lipid membrane and forms lipid-mediated interactions with the neighboring subunit.

We also find lipid moieties, structured to various degrees, in the crevices between the scaffold and transport domains (Figure 5a). Of these, the most notable one is inserted between the N-terminal arm of HP2 and the scaffold TM4a (Lipid_Out Figure 5a,b). Interestingly, we observe lipids at almost the same location in the Cryo-EM structures of the outward- (Huang et al., 2020) and inward-facing L-asp-bound transporters (Figure 5b). The lipid packs similarly against TM4a in the OFS and IFS but interacts differently with HP2: near the tip and the extracellular base, respectively. It is not yet clear whether during the outward-to-inward transition, as HP2 slides past TM4a, the lipid is temporarily displaced or disordered. Interestingly, HP2 opening in the OFS, as seen in Glt_Ph^OFS-TBOA, and the IFS requires displacement of Lipid_Out. Thus, the lipid molecules at this site could modulate gating and the translocation dynamics, affecting both substrate affinity and transport rate. In Glt_Ph^IFS-TFB-TBOA and Glt_Ph^IFS-Apo-open structures, the transport domain leans away from the scaffold far enough to open a window between the two domains that connects the interior of the bilayer to the solvent-filled crevice on the cytoplasmic side of the transporter (Figure 5c). We observe excess densities in the opening, suggesting that lipids enter the space at a position structurally symmetric to Lipid_Out (Lipid_Window, Figure 5c).

Perhaps most strikingly, we observe nanodisc distortions correlated to positions of transport domains (Figure 5d,e, Video 3). The nanodisc is nearly flat in the Glt_Ph^OFS-TBOA structure, where the hydrophobic regions of the transport domain and the scaffold are aligned. In Glt_Ph^IFS-Asp, the transport domain forms the sharpest angle to the membrane normal (Figure 1a), and its hydrophobic region descends the furthest toward the cytoplasm. The resulting hydrophobic mismatch between the scaffold and transport domains leads to membrane bending to accommodate both, as suggested by recent computational studies (Zhou et al., 2019) and studies of Glt_Tk (Arkhipova et al., 2020). At the protein periphery, the membrane deformation at the transport domain reaches ~ 8 Å shift toward the cytoplasm, observable from both sides of the nanodisc (Figure 5d,e, Video 3). In contrast, when the inward-facing transport domains swing out, their hydrophobic regions are closer to the extracellular side, and the membrane is less bent. In an extreme case of Glt_Ph^IFS-TFB-TBOA structure, the membrane bends outward, particularly when viewed from the cytoplasmic side (Figure 5d,e, Video 3). It is unclear whether the nanodisc restricts how far the transport domains swing in the Glt_Ph^IFS-TFB-TBOA structure. Indeed, we observe interactions between the domains and the MSP1E3 lipoprotein, suggesting the size of the nanodiscs might be limiting. Notably, structures of glutamate transporter homologs determined in detergent solutions featured similar positions of the domains (Garaeva et al., 2019; Akyuz et al., 2015).

The fully functional seven-histidine mutant of Glt_Ph that has been used in previous studies and that is referred to as wildtype (WT) for brevity, and the K55C/C321A/A364C Glt_Ph mutant were expressed as C-terminal His₈ fusions and purified as described previously (Yernool et al., 2004). Briefly, the plasmids were transformed into E. coli DH10-B cells (Invitrogen). Cells were grown in LB media supplemented with 0.2 mg/L of ampicillin (Goldbio) at 37°C until OD₆₀₀ of 1.0. Protein expression was induced by adding 0.2% arabinose (Goldbio) for 3 hr at 37°C. The cells were harvested by centrifugation and re-suspended in 20 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM L-asp, 1 mM EDTA. The suspended cells were broken using Emulsiflex C3 high pressure homogenizer (Avestin Inc) in the presence of 0.5 mg/mL lysozyme (Goldbio) and 1 mM phenylmethanesulfonyl fluoride (PMSF, MP Biomedicals). After centrifugation for 15 min at 5000 g at 4°C to remove the debris, membranes were pelleted by centrifugation at 125,000 g for 60 min. The membranes were homogenized in 20 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM L-asp, 10 mM EDTA, 10% sucrose and pelleted again by centrifugation at 125,000 g for 60 min. The washed membranes were collected and solubilized in Buffer A, containing 20 mM Hepes, pH7.4, 200 mM NaCl, 1 mM L-asp, supplemented with 40 mM n-dodecyl-β-D-maltopyranoside (DDM, Anatrace, Inc) at 8 mL per gram of membranes for 2 hr at 4°C. The mixture was clarified by ultracentrifugation for 60 min at 125,000 g, the supernatant was incubated with Ni-NTA resin (Qiagen) pre-equilibrated in buffer A with gentle shaking for 2 hr at 4°C. The resin was washed with 5 volumes of Buffer A with 1 mM DDM and 25 mM imidazole, the protein was eluted in the same buffer containing 250 mM imidazole. The eluted protein was concentrated using concentrators with 100 kDa MW cutoff (Amicon). The (His)₈-tag was cleaved by thrombin (Sigma) using 20 U per 1 mg Glt_Ph in the presence of 5 mM CaCl₂ at room temperature overnight. The reaction was stopped by addition of 10 mM EDTA and 1 mM PMSF. For the WT Glt_Ph, the protein was further purified by size exclusion chromatography (SEC) in buffer A and 1 mM DDM. The eluted protein was concentrated and used immediately for nanodisc reconstitution. After affinity chromatography and (His)₈-tag removal, prior to crosslinking, the K55C/C321A/A364C mutant protein was reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 1 hr. Protein was then exchanged into buffer A with 1 mM DDM, using filters (Amico, Inc) with a molecular weight cutoff of 100 kDa. Reduced K55C/C321A/A364C Glt_Ph at concentrations below 1 mg/mL was incubated with 10-fold molar excess of HgCl₂ for 15 min at room temperature. The protein was concentrated to under 1 ml and purified by SEC in buffer A supplemented with 1 mM DDM. The elution peak fractions were collected and concentrated. The protein concentration was determined by UV absorbance at 280 nm using extinction coefficient of 57,400 M⁻¹ cm⁻¹ and MW of 44.7 kDa. To check availability of free thiols after crosslinking, proteins were incubated with 5-fold molar excess of fluoroscein-5-maleimide (F5M). Fluorescent F5M-labeled proteins were imaged on SDS-PAGE under blue illumination and stained with Coomassie blue.

Membrane scaffold protein MSP1E3 (Denisov et al., 2004) was expressed and purified from E. coli and Glt_Ph was reconstituted into lipid nanodiscs as previously described, with modifications (Ritchie et al., 2009). Briefly, E. coli polar lipid extract and egg phosphatidylcholine in chloroform (Avanti) were mixed at 3:1 (w:w) ratio and dried on rotary evaporator and under vacuum overnight. The dried lipid film was resuspended in buffer containing 20 mM Hepes/Tris, pH 7.4, 200 mM NaCl, 1 mM L-asp and 80 mM DDM by 10 freeze/thaw cycles resulting in 20 mM lipid stock. The purified Glt_Ph protein in DDM was mixed with MSP1E3 and lipid stock at 0.75:1:50 molar ratio at the final lipid concentration of 5 mM and incubated at 21°C for 30 min. Biobeads SM2 (Bio-Rad) were added to one third of the reaction volume and the mixture was incubated at 21°C for 2 hr on a rotator. Biobeads were replaced and incubated at 4°C overnight. The sample containing Glt_Ph^IFS reconstituted into the nanodiscs in the presence of 1 mM L-asp was cleared by centrifugation at 100,000 g and purified by SEC using a Superose 6 Increase 10/300 GL column (GE Lifesciences) pre-equilibrated with buffer containing 20 mM Hepes/Tris, pH 7.4, 200 mM NaCl and 1 mM L-asp. The peak fractions corresponding to Glt_Ph^IFS-containing nanodiscs were collected for Cryo-EM imaging. To prepare substrate-free WT Glt_Ph and Glt_Ph^IFS in nanodiscs, the reconstitution mixtures were cleared by centrifugation at 100,000 g, diluted with 10 x volume of buffer containing 20 mM Hepes/Tris, pH 7.4, and 50 mM choline chloride, and concentrated using 100 kDa cutoff concentrator. After repeating the procedure twice, substrate-free transporters in nanodiscs were purified by SEC in the same buffer. The peak fractions were collected and immediately supplemented with buffers containing 200 mM NaCl and 10 mM DL-TBOA, 200 mM NaCl and 10 mM TFB-TBOA, or 200 mM NaCl. The presence of the MSP1E3 and Glt_Ph proteins in the samples was confirmed by SDS-PAGE. Negative staining electron microscopy was used to confirm the formation and the homogeneity of the nanodisc samples.

To prepare cryo-grids, 3.5 μL of Glt_Ph-containing nanodiscs (7 mg/mL) supplemented with 1.5 mM fluorinated Fos-Choline-8 (Anatrace) were applied to a glow-discharged UltrAuFoil R1.2/1.3 300-mesh gold grid (Quantifoil) and incubated for 20 s under 100% humidity at 15°C. Grids were blotted for 2 s and plunge frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific). For the WT Glt_Ph in the presence of DL-TBOA (Glt_Ph^OFS-TBOA), Glt_Ph^IFS in the presence of TFB-TBOA (Glt_Ph^IFS-TFB-TBOA), and Glt_Ph^IFS in the presence of 200 mM Na⁺ ions only (Glt_Ph^IFS-NaCl), the Cryo-EM imaging data were acquired using a Titan Krios microscope (Thermo Fisher Scientific) at New York Structural Biology Center operated at 300 kV with a K2 Summit detector with a calibrated pixel size of 1.07325 Å/pixel. A total dose of 68.55 e^–/Ų (Glt_Ph^OFS-TBOA, Glt_Ph^IFS-NaCl), or 68.70 e^–/Ų (Glt_Ph^IFS-TFB-TBOA) distributed over 45 frames (1.52 e^–/ Ų/frame) was used with an exposure time of 9 s (200 ms/frame) and a defocus range of −1.5 μm to −2.5 μm. For Glt_Ph^IFS in the presence of DL-TBOA (Glt_Ph^IFS-TBOA), Cryo-EM imaging data were acquired on a Titan Krios microscope at New York Structural Biology Center operated at 300 kV with a K2 Summit detector with a calibrated pixel size of 1.07325 Å/pixel. A total dose of 69.70 e^–/Ų distributed over 50 frames (1.52 e^–/ Ų/frame) was used with an exposure time of 10 s (200 ms/frame) and a defocus range of −1.5 μm to −2.5 μm. For the Glt_Ph^IFS in the presence of L-asp (Glt_Ph^IFS-Asp), micrographs were acquired on a Titan Krios microscope at New York Structural Biology Center operated at 300 kV with a K2 Summit detector, using a slid width of 20 eV on a GIF Quantum energy filter with a calibrated pixel size of 1.0605 Å/pixel. A total dose of 69.30 e^–/Ų distributed over 45 frames (1.54 e^–/ Ų/frame) was used with an exposure time of 9 s (200 ms/frame) and defocus range of −1.5 μm to −2.5 μm. For all samples, automated data collection was carried out using Leginon (Suloway et al., 2005).

The frame stacks were motion corrected using MotionCorr2 (Zheng et al., 2017) and contrast transfer function (CTF) estimation was performed using CTFFIND4 (Rohou and Grigorieff, 2015). All further processing steps were done using RELION 3.0 (Glt_Ph^IFS-Asp, Glt_Ph^IFS-TBOA, Glt_Ph^IFS-TFB-TBOA, Glt_Ph^IFS-NaCl) or Relion 3.1 (Glt_Ph^IFS-TBOA) unless otherwise indicated (Zivanov et al., 2018). DoGpicker (Voss et al., 2009) as part of the Appion processing package (Lander et al., 2009) was used for reference-free particle picking. Picked particles were then extracted and subjected to 2D classification to generate 2D class-averages which were used as templates for automated particle picking in Relion. The particles were extracted using a box size of 275 Å with 2x binning and subjected to 2 rounds of 2D classification ignoring CTFs until the first peak.

For Glt_Ph^IFS-Asp, Glt_Ph^IFS-TBOA, Glt_Ph^IFS-TFB-TBOA, and for the Glt_Ph^OFS-TBOA, particles selected from 2D classification were re-extracted without binning and further classified into six classes without enforcing symmetry using initial models generated in CryoSPARC (Punjani et al., 2017) and filtered to 40 Å. Particles from the best classes showing trimeric transporter arrangements were subjected to 3D refinement applying C3 symmetry. After conversion, the refinement was continued with a mask excluding the nanodisc. To further improve the resolution of the maps, the particles after 3D refinement were subject to an additional round of 3D classification without alignment with C3 symmetry applying a mask to exclude the nanodisc. Particles from the best class were subjected to further masked refinement and CTF refinement. A masked refinement following CTF refinement yielded final maps with the following resolution: 3.05 Å (Glt_Ph^IFS-Asp), 3.71 Å (Glt_Ph^IFS-TFB-TBOA), 3.39 Å (Glt_Ph^IFS-TBOA), 3.66 Å (Glt_Ph^OFS-TBOA). The resolution limits of the refined maps were assessed using Relion postprocessing and gold standard FSC value 0.143 using masks that excluded the nanodiscs. To search for potential conformational heterogeneity, we also processed these datasets with no symmetry applied at any stage of data processing (C1). The obtained C1 maps showed slightly lower resolution but no detectable difference when compared to the results from the C3 refinement. We also processed all datasets with symmetry expansion (C3) followed by focused 3D classification on one Glt_Ph subunit (explained in detail for Glt_Ph^IFS-Na data processing below) and did not find additional conformations. The cryo-EM map of Glt_Ph^IFS-TBOA was processed using the RESOLVE density modification program implemented in Phenix, which improved the overall estimated resolution by 0.01 Å and enabled slightly better visualization of the density of the bound TBOA benzyl group (Terwilliger et al., 2020; Afonine et al., 2010).

During processing of the data for Glt_Ph^IFS-NaCl, 529,155 particles selected from 2D classification were re-extracted without binning and were subjected to 3D classification with K = 1 and no symmetry applied, using Glt_Ph^IFS-Asp map as the initial model. The same particles were subject to 3D refinement with C3 symmetry. After conversion, the refinement was continued with a mask to exclude the nanodisc, resulting in a 3.56 Å resolution map. To probe for conformational heterogeneity, we performed symmetry expansion implemented in Relion (Scheres, 2016). 1,587,465 protein subunits were rotated to the same position and subjected to a focused 3D classification without alignment with T = 40 into 10 classes. The local mask was generated using Chain A of PDB model 3KBC (Reyes et al., 2009) and included only densities from one subunit of the reference map. Two different conformations were observed. From the 10 classes, five classes showed a conformation identified as Glt_Ph^IFS-Na and five classes showed a different conformation identified as Glt_Ph^IFS-Apo-open. The best Glt_Ph^IFS-Na class (191,349 particles), which contained 12% of the symmetry expanded protomers and the best Glt_Ph^IFS-Apo-open class (148,582 particles), which contained 9% of the symmetry expanded particles, were separately subjected to a final focused 3D refinement with C1 using a mask to exclude the nanodisc. The local angular searches in this refinement were conducted only around the expanded set of orientations to prevent contributions from the neighbor subunits in the same particle. The resulting maps were postprocessed in Relion using the same mask as in 3D classification after symmetry expansion. The final resolution at gold standard FSC value 0.143 was estimated as 3.52 Å for the Glt_Ph^IFS-Apo-open map and 3.66 Å for Glt_Ph^IFS-Na map. Local resolution variations were estimated using ResMap (Kucukelbir et al., 2014). After symmetry expansion with C3, we also tried to first subtract the density outside of one Glt_Ph subunit and then perform 3D classification without alignment on the subtracted particles. The signal subtraction did not further improve the 3D classification and the 3D refinement.

For atomic model building from Glt_Ph^IFS-Asp, Glt_Ph^IFS-TBOA, and Glt_Ph^IFS-TFB-TBOA maps, crystal structure of Glt_Ph in the IFS (PDB code 3KBC) (Reyes et al., 2009) was docked into the density maps using UCSF Chimera (Pettersen et al., 2004). For the WT Glt_Ph^OFS-TBOA, crystal structure of Glt_Ph in the OFS (PDB code 2NWW) (Boudker et al., 2007) was docked into the density. For Glt_Ph^IFS-Na or Glt_Ph^IFS-Apo-open, one subunit of 3KBC was docked into the density. After the first rounds of the real-space refinement using Phenix (Afonine et al., 2010), miss-aligned regions were manually rebuilt and missing side chains and residues were added in COOT (Emsley et al., 2010). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) was used as a model lipid and placed into the excess densities which resembled lipid molecules. The acyl chains or ethanolamine heads were truncated to fit the visible densities. Models were iteratively refined applying secondary structure restraints and validated using Molprobity (Chen et al., 2010). For further cross validation and to check for overfitting, all atoms of each model were randomly displaced by 0.3 Å and each resulting model was refined against the first half-map obtained from processing. FSC between the refined models and the half-maps used during the refinement were calculated and compared to the FSC between the refined models and the other half-maps. In addition, the FSC between the refined model and sum of both half-maps was calculated. The resulting FSC curves were similar showing no evidence of overfitting.

For ITC experiments, Glt_Ph K55C/C321A/A364C proteins were purified by affinity chromatography as above. After (His)₈-tag removal, prior to crosslinking, the K55C/C321A/A364C protein was reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 1 hr. Protein was then exchanged into buffer A with 1 mM DDM using filters (Amico, Inc) with a molecular weight cutoff of 100 kDa. Reduced K55C/C321A/A364C Glt_Ph at concentrations below 1 mg/mL was incubated with 10-fold molar excess of HgCl₂ for 15 min at room temperature. The protein was concentrated, diluted with 10 x volume of substrate-free buffer containing 20 mM Hepes/Tris, pH 7.4, 50 mM choline chloride and 1 mM DDM, and re-concentrated. After repeating the procedure twice, the protein was purified by SEC in the same buffer. Protein samples at 40 μM in substrate-free buffer supplemented with 200 mM NaCl, were loaded into the reaction cell of an Affinity ITC (TA Instruments, Inc). The injection syringe was loaded with a solution containing 20 mM Hepes/Tris, pH 7.4, 50 mM choline chloride, 200 mM NaCl, 400 μM TFB-TBOA or DL-TBOA. Titrant aliquots of 2 μL were injected every 5 min at 15°C. Binding isotherms were fitted to independent binding site model using NanoAnalyze software (TA Instruments, Inc).