Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.

Production and purification of MSP2N2 The gene encoding the membrane scaffold protein MSP2N2 with the His 7 -tag at the N-terminus cloned in the pET28a plasmid (Addgene) was expressed in E.coli BL21(DE3) cells (New England Biolabs). Cells were cultivated in LB medium in the presence of 50 µg/mL of kanamycin at 37 °C, 200 r.p.m. until the OD 600 reached 0.8. Then expression was induced by addition of 0.1 mM IPTG and after 3.5 h cells were harvested by centrifugation (15 min, 6270 × g , 4 °C). Cells were resuspended in 40 mM Tris-HCl, pH 7.8, 300 mM NaCl and stored at −80 °C. For purification cells were thawed and supplemented with 100 µg/mL DNase A, 1 mM MgSO 4 and 1 mM PMSF. Cells were passed through a cell disrupter at 4 °C and 25 kPsi (Constant Systems Ltd. Daventry UK). One percent of TritonX-100 was added to the lysate followed by stirring for 10 min at room temperature. After centrifugation at 30,000 × g , 30 min, 4 °C supernatant was supplemented with 20 mM Imidazole, pH 8.0, and MSP2N2 was purified using Ni-NTA resin. MSP2N2 was eluted in 40 mM Tris-HCl, pH 8.0, 300 mM NaCl, 500 mM Imidazole. To cleave off the His-tag, protein fractions were pooled, supplemented with 1 mM Na-EDTA and 1:40 w/w TEV (Sigma-Aldrich) and dialyzed against 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT overnight at 4 °C using a Servapor® dialysis tubing. The cleaved protein was loaded onto Ni-NTA resin and collected in the flow-through fraction using 50 mM Tris-HCl, pH 8.0, 100 mM NaCl. Protein fractions were stored at −80 °C. Production and purification of apo Glt Tk For production of C-terminally His 8 -tagged Glt Tk , E. coli MC1061 containing a pBAD24 derived plasmid were grown in LB medium with 100 µg/mL ampicillin at 37 °C, 200 r.p.m. When the OD 600 reached 0.8 expression was induced with 0.05% l -arabinose for 3 h. Cells were harvested by centrifugation (15 min, 7400 × g , 4 °C) and resuspended in 20 mM Tris-HCl, pH 8.0. After breaking cells (25 kPsi, 5 °C, Constant Systems Ltd. Daventry UK) the membrane fraction was collected by ultracentrifugation of the supernatant (90 min, 193,360 × g , 4 °C), resuspended in 20 mM Tris-HCl, pH 8.0, and stored at −80 °C. To obtain apo -Glt Tk Na + was omitted from all buffers. An aliquot of membrane vesicles representing ~1.2 g cells was solubilized in 50 mM Tris-HCl, pH 8.0, 300 mM KCl, 1% n-dodecyl-β- d -maltoside (DDM) for 1 h at 4 °C. After ultracentrifugation (30 min, 265,000 × g , 4 °C) the supernatant was incubated with Ni-Sepharose resin (GE Healthcare) for 1 h at 4 °C. The column was washed with 50 mM Tris HCl, pH 8.0, 300 mM KCl, 0.15% n-decyl-β- d -maltoside (DM), 60 mM Imidazole, pH 8.0, and the protein was eluted with the same buffer containing 500 mM Imidazole. Glt Tk was further purified by size exclusion chromatography on a Superdex 200 10/300 gel-filtration column (GE Healthcare) in 10 mM Hepes KOH, pH 8.0, 100 mM KCl, 0.15% DM.

Nanodiscs reconstitution and cryo-EM sample preparation

Twenty milligram per milliliter of liposomes containing E. coli polar lipids and egg PC (w/w 3:1, Avanti), were solubilized by adding 30 mM DDM followed by 1 min vortexing and 3 h incubation at 4 °C while nutating. Freshly prepared apo Glt Tk, cleaved MSP2N2 and solubilized lipids were mixed in 3:5:100 molar ratio (considering a single protein chain) and incubated at 4 °C for 90 min while nutating. To remove the detergent BioBeads (Bio-Rad) were added and the mixture was incubated overnight at 4 °C. After removing BioBeads using a syringe the solution was supplied with 15 mM Imidazole, pH 8.0, and Ni-NTA resin, equilibrated with 50 mM Tris-HCl, pH 8.0, 100 mM KCl to remove empty nanodiscs. After 1 h incubation at 4 °C on a rocking platform resin was washed with 50 mM Tris-HCl, pH 8.0, 300 mM KCl, 30 mM Imidazole, pH 8.0 and nanodiscs were eluted with 500 mM Imidazole in the same buffer. The sample was centrifuged (10 min, 20,000 × g , 4 °C) and applied to Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl. Fractions with nanodiscs were concentrated using VivaSpin® 500 MWCO 50 kDa concentrators (Sartorius) to 1–4 mg/mL. The sample was supplemented with NaCl to 300 mM (Na + -only state) and additionally with l -aspartate (Sigma-Aldrich) at the molar ratios Glt Tk : l -aspartate equal to 3:1 for unsaturated state (9 µM of both nanodiscs and l -aspartate) or 1:3 for saturated state (5.6 µM nanodiscs and 50 µM l -aspartate) or with DL-TBOA (4.5 µM nanodiscs and 120 µM DL-TBOA). After mixing the sample was incubated for 30 min on ice. The sample at 0.5–1 mg/mL was applied onto freshly glow-discharged Quantifoil grids (Au R1.2/1.3, 300 mesh) at 22 °C and 100% humidity and plunged-frozen in liquid ethane. The Cryo-EM data were collected using 200 keV Talos Arctica microscope (Thermo Fisher).

Cryo-EM image processing

Motion correction, CTF estimation, template-based picking, 2D classification, Ab initio volume generation and non-uniform 3D refinement (without symmetry applied if not stated otherwise) were performed using cryoSPARC 47 (Supplementary Fig. 1 ). Maps were sharpened using Autosharpen Map procedure in Phenix 48 . The sharpened maps were used for the manual model building using Coot 49 and refinement of the coordinates was performed in realspace refine module of Phenix 50 , 51 . Visualization and structure interpretation were carried out in UCSF Chimera 52 , 53 and PyMol (Schrödinger, LLC). For the data obtained using protein in aspartate-free conditions, 1288 micrographs were selected for the processing after motion correction and CTF estimation. The template for particle picking was generated from 100 manually picked particles. Template-based picking identified 1,193,046 particles. Subsequent 2D classification reduced the number of particles to 334,624 and subsequently 180,608 (49%) particles were left in the selected ab initio class. Final non-uniform 3D refinement resulted in a 3.22 Å map, where one protomer was oriented up ( apo state) and two protomers were inward oriented. Other particles from ab initio volume generation were combined and refined, providing a low-resolution map of 4.82 Å (2 out:1 in). For the data obtained using protein in substrate unsaturated conditions, 956 micrographs were selected after motion correction and CTF estimation. 348,173 particles were picked and 149,715 particles were left after subsequent 2D classification. Ab initio volume generation provided three classes of 72,313 (48%), 59,666 (40%), and 17,736 (12%) particles. Two big classes were used for non-uniform 3D refinement without applying a symmetry and resulted in maps of 3.38 Å (2 out:1 in) and 3.39 Å (2 in:1 out). For the data obtained using protein in substrate saturated conditions, 541 micrographs were selected after motion correction and CTF estimation. 444,955 particles were picked and 92,538 particles were left after subsequent 2D classification. Ab initio volume generation provided with one class of 65,762 (71%) particles that was used for non-uniform 3D refinement without applying a symmetry. The resulting map of 4.0 Å resolution revealed symmetrical arrangement of the three protomers, therefore C3 symmetry was applied to generate a final 3.41 Å map. For the data obtained using protein in TBOA-inhibited conditions, 1,675 micrographs were selected after motion correction and CTF estimation and 578,728 particles were picked. Following 2D classification a set of 228,131 particles was left. After ab initio volume generation one class of 132,917 (58%) particles was chosen for further non-uniform 3D refinement without applying a symmetry. The resulting map of 3.47 Å resolution revealed an asymmetric state with two fully-outward and one inward oriented protomers. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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