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Published in final edited form as: Structure. 2026 Aug 31;34(10):1405–1413.e2. doi: 10.1016/j.str.2026.08.003

Structures of the human sodium-citrate cotransporter NaCT with and without substrates

David B Sauer 1,#,*, Jinmei Song 1, Jennifer J Marden 1, Bing Wang 2, Kate Sowerby 1, Joseph C Sudar 1, William J Rice 1,2,*, Da-Neng Wang 1,$,*
PMCID: PMC13637752  NIHMSID: NIHMS2204263  PMID: 42673948

Summary

The human sodium-citrate cotransporter NaCT imports various tri- and dicarboxylates into the cell as TCA cycle intermediates. This substrate uptake process is driven by an inward sodium gradient. The protein is a member of the Divalent Anion-Sodium Symporter (DASS) family. Whereas extensive biochemical and structural studies have been carried out for NaCT, how the substrate binding and translocation is coupled to the sodium gradient remains unclear. Here using single particle cryo-electron microscopy, we determined the structures of the human NaCT protein in three states: sodium-free, in the presence of sodium, and bound to sodium and a substrate mimicking inhibitor. These structures suggest a simultaneous binding mechanism for sodium-substrate coupling, distinct from the sequential binding, conformational selection mechanism previously observed for the bacterial DASS protein VcINDY.

Keywords: Sodium-substrate coupling, citrate, SLC13A5, cotransporter, transporter dynamics

In Brief

Uptake of citrate by human NaCT is driven by a sodium gradient. Its sodium-substrate coupling mechanism is unclear. Sauer et al. determined cryo-EM structures of the protein in apo, in sodium, and in sodium and substrate mimicking inhibitor. These structures suggest a simultaneous binding mechanism of coupling in the transporter.

Graphical Abstract

graphic file with name nihms-2204263-f0005.webp

1. Introduction

Citrate plays several important roles in the cell as both an energy source and a major metabolite in the TCA cycle. Not only is the metabolite a major precursor for de novo fatty acid biosynthesis 1, its concentration in the cytosol upregulates this synthesis pathway by directly binding to the enzyme at the rate-limiting step of the pathway 2,3. In the nervous system, where the citrate levels can be as high as 0.4 mM in the cerebrospinal fluid 4, the imported tricarboxylate acts as a precursor for neurotransmitter synthesis and can regulate neuronal excitability through the NMDA glutamate receptors 5–7. Disrupting citrate catabolism and intracellular citrate accumulation can lead to stress responses and impairs cell fitness 8. Therefore, it is critical that the cytosolic levels of citrate are carefully controlled.

The cell has two major sources for obtaining cytosolic citrate, export from the mitochondria and import from the extracellular milieu across the plasma membrane. The plasma membrane transporter NaCT (SLC13A5) couples the import of citrate and other di- and tricarboxylates from the extracellular environment to the co-transport of four sodium ions 9,10. Various disease phenotypes have been associated with NaCT mutants, including dental hypoplasia and early onset epilepsy 11–14. The disease-associated NaCT mutations either reduce the protein’s folding and stability, directly abolish substrate binding, or hinder conformational changes required for translocating the substrate across the membrane 13,15. Multiple inhibitors have been developed as starting points or tool molecules for the therapeutic targeting NaCT to treat obesity, kidney diseases and epilepsy 16–19, including the substrate mimicking PF2 20,21.

NaCT is a member of the solute carrier 13 gene family (SLC13) of anion transporters 22–24. Other members include the sodium-dicarboxylate cotransporters NaDC1 and NaDC3. The mammalian SLC13 proteins are part of the evolutionarily conserved Divalent Anion-Sodium Symporter (DASS) family, with the best characterized member, VcINDY 25–28, serving as a prototype for the sodium-coupled anion transport mechanism of the entire family 29–32. Over the past few years, significant progress has been made in the mechanistic understanding of the SLC13/DASS proteins, particularly through structure determination 28. DASS transporters form homodimers, where each protomer is composed of a scaffold domain and a mobile transport domain, with the substrate and sodium sites located at the middle of the transport domain. The substrate binding site is sandwiched between two sodium sites, Na1 and Na2. Such proximity of the anionic substrate with the sodium ions allows for charge compensation, helping to reduce the energy barrier for substrate translocation across the membrane 33–35. For NaDC1 and NaDC3, structures of both the inward-facing (Ci) and outward-facing (Co) conformations have been observed 30,31, whereas the NaCT structure has only been determined in the inward-facing conformation and Na+-substrate or inhibitor bound states 29,31. Such structural information, along with cross-linking experiments, molecular dynamics simulations and single-molecule Förster resonance energy transfer measurements 27,36,37 have demonstrated that these SLC13 proteins and their bacterial homologs operate via an elevator mechanism for substrate transport 38,39.

Despite such progress, one important mechanistic question for NaCT remains unaddressed, namely: How is substrate transport coupled to sodium binding? The coupling mechanism for substrate and co-substrate in most secondary membrane transporters is poorly understood 40,41. Early whole cell transport kinetics and patch-clamp measurements indicate that rabbit NaDC1 employs a sequential binding scheme, with sodium binding first, followed by the dicarboxylate substrate 42,43. In this mechanism, it is proposed that the substrate release process mirrors the binding process, wherein the dicarboxylate is released first, followed by the sodium ions. Transport activity and substrate binding measurements showed that such a sequential binding scheme applies to bacterial DASS transporters as well 44–46. Notably, the only DASS protein for which the sodium-substrate coupling mechanism has been extensively characterized is the dicarboxylate transporter from Vibrio cholerae, VcINDY. Cryo-EM structure determination of the transporter in different states along with site-directed labelling assays showed that sequential binding is driven by a sodium-induced conformational selection mechanism 27,35. In this mechanism, the binding of sodium ions at the Na1 and Na2 sites selects from an ensemble of loops and rigidifies them to a single configuration that can subsequently coordinate the dicarboxylate substrate in VcINDY 35.

In addition to citrate, NaCT also transports various dicarboxylates such as succinate and α-ketoglutarate. The transporter binds two sodium ions at the canonical Na1 and Na2 sites of DASS transporters, proximal to the citrate site 29. However, it is unknown whether NaCT also follows the same sequential binding, conformational selection mechanism for sodium-substrate coupling. To address this question, we have determined the cryo-EM reconstructions of human NaCT in the absence of sodium, in the presence of sodium, and in sodium and a substrate-mimicking inhibitor. These structures provide high-resolution detail of NaCT’s structure in apo, sodium-saturated, and sodium- and dicarboxylate-bound states, and provide an understanding of the protein’s sodium and substrate coupling mechanism.

2. Results

Cryo-EM can yield structures of a transporter protein in various substrate-bound states 31,47. Applying VcINDY’s conformational selection mechanism for sequential binding 35 to NaCT predicts that (1) in the absence of sodium, the human protein apo structure is flexible at the Na1 and Na2 sites; (2) binding of sodium stabilizes a discrete, rigid structure that can coordinate the di-/tricarboxylate substrate (S). (3) As a consequence of this mechanism at the Na1/Na2/S sites, the NaCT-Na+ structure will differ from the apo state but will resemble the NaCT-Na+-S state. To examine these predictions, we need the structures of NaCT-Na+-S, NaCT-Na+, and NaCT-apo.

2.1. NaCT-Na+-PF2 structure

In our previous studies, structures of NaCT-Na+-S have been determined 29,31 in the presence of 100 mM NaCl, together with one of the four substrates, citrate, succinate, PF2 (PF-06649298) and PF4a (PF-067611281), with the latter two being substrates that enter the cell via NaCT, bind from the cytosol and become competitive inhibitors 20,21. Under all conditions tested, the transporter dimer adopts a symmetric, inward-facing Ci-Ci conformation. However, precise analysis of the protein structure and ion binding in these structures is challenged due to the weak affinity of DASS transporters for sodium (K0.5 ~70 mM) 48–51, which would result in low occupancy for sodium ions at 100 mM within the protein. The density for sodium is likely diminished further by the 20 mM LiCl included in the cryo-EM sample preparation, where this lighter ion thermostabilizes the protein 29 but gives a weaker signal in the Coulombic potential map 52.

To resolve this ambiguity in sodium visibility in NaCT maps, we purified NaCT in the presence of both 300 mM NaCl and 100 μM PF2, but in the absence of LiCl. This sodium concentration of 300 mM was chosen to balance the desire for high occupancy with the need to avoid any salting-out effects on the protein in solution and to minimize background noise in the electron micrographs 27,53,54. PF2 was chosen over citrate in the structure determination for higher affinity and occupancy, with the protein’s substrate- and inhibitor-bound structures effectively identical 29. The detergent-purified protein was exchanged into the amphipol polymer and cryo-EM grids were frozen. The NaCT particles on the grids displayed preferred orientation problems as was observed with previous samples prepared in 100 mM NaCl, which we once again overcame by collecting electron micrographs at 0 and 40° tilts 29,55. Processing the merged datasets allowed us to solve the NaCT structure at an overall resolution of 2.53 Å (Figures 1A & B, Figure S1). The protein adopted a Ci-Ci dimer structure (Figure 1C), with a clear density for the PF2 inhibitor (Figure 1D). Furthermore, non-protein density was observed at the Na1 and Na2 sites which are consistent with bound sodium ions, within the resolution limit of the map, confirming that NaCT was captured in the Ci-Na+-PF2 state (Figure 1E & F). The overall structure is similar to that previously solved at 3.12 Å resolution in 100 mM NaCl, 20 mM LiCl and 100 μM PF2 29 (R.M.S.D. = 0.40 Å). Nevertheless, the higher experimental resolution and clearer densities at the PF2, Na1, and Na2 binding sites enabled us to build a more accurate structural model for the protein and its bound cations and substrate (Table S1). This new PF2-bound structure is very similar to the one previously-determined in citrate 29 (R.M.S.D. = 0.79 Å), and we thus conclude it represents the substrate-bound NaCT-Na+-S state.

Figure 1. High-resolution structure of sodium and PF2-bound NaCT.

Figure 1.

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(A) Columbic potential map of NaCT determined to 2.53 Å resolution. Atomic model of NaCT viewed from (B) within the membrane and (C) outside the cell. (D) The PF2-binding site structure with Columbic potential map is shown in mesh. The scaffold and transport domains are colored light green and light pink, respectively. The PF2 substrate-mimicking inhibitor is shown in yellow. The (E) Na1 and (F) Na2 binding sites with the cryo-EM map shown as mesh and contoured at 10 σ.

2.2. Structures of sodium-bound and apo NaCT

To identify structural changes in NaCT through its reaction cycle, we next determined the structures of NaCT in the absence of substrate, or in the absence of both substrate and sodium, to capture the NaCT-Na+ and NaCT-apo states, respectively. The NaCT-Na+ sample was prepared in the presence of 300 mM NaCl, whereas the apo sample was purified in 100 mM choline chloride (ChCl). Choline has been shown to be an organic cation that can maintain the integrity and stability of Na+ channels and Na+-driven transporters during purification 35,56,57. As with the previous sample in NaCl and PF2, NaCT particles on both the 300 mM NaCl and 100 mM ChCl grids exhibited preferred orientations. Nevertheless, using data collection from tilted grids 29,55, we were able to obtain 3D maps and protein models of the protein at overall isotropic resolutions of 2.51 Å and 2.49 Å (Table S1, Figures S2&S3), for sodium-saturated and sodium-free samples, respectively (Figures 2A&B, 2E&F). No density was seen at the citrate, Na1 or Na2 sites for NaCT in 100 mM ChCl (Figures 2G&H), confirming that we have captured the protein in an apo state.

Figure 2. Structure of NaCT’s Ci-Na+ and Ci-apo states.

Figure 2.

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(A) The Columbic potential map and (B) atomic model of NaCT-Na+. (C) The Na1 and (D) Na2 binding sites for the NaCT-Na+ state with the cryo-EM map shown as mesh and contoured at 10 σ. (E) The Columbic potential map and (F) atomic model of NaCT-apo. (G) The Na1 and (H) Na2 binding sites for the NaCT-apo state with the cryo-EM map shown as mesh and contoured at 10 σ.

Remarkably, we did not observe any density at the Na1 and Na2 sites for NaCT in 300mM NaCl (Figures 2C&D), and the protein density near the Na2 site was of poor quality in both conditions. The absence of cation densities at the Na1and Na2 sites in the sodium-only condition and the increased protein mobility near Na2, are both marked differences from VcINDY in equivalent conditions 35 and suggest a different cation-substrate coupling mechanism for NaCT.

Structures of sodium-saturated and apo NaCT both adopted a symmetric, inward-facing conformation, termed Ci-Na+ and Ci-apo, respectively. Notably, while the Ci-Na+ and Ci-apo structures were essentially identical (R.M.S.D. = 0.48 Å) (Figure 3A), there were marked changes between Ci-Na+ and Ci-Na+-PF2 structures (R.M.S.D. = 1.1 Å). This is most pronounced on the horizontal helices H4c and H6b (Figure 3A), and apparent movements of HPin, and TM10b on the cytoplasmic face of the transport domain (Figure 3B), necessitating a detailed exploration of how PF2 release triggers these subtle structural movements.

Figure 3. Carboxylate release triggers conformational changes in the NaCT transport domain.

Figure 3.

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A) Movements in the NaCT transport domain and arm helices H4c and H6b upon PF2 release. Ci-Na+-PF2, Ci-Na+, and Ci-apo states are colored in blue, green, and pink, respectively. PF2 release triggers movements in (B) HPin and TM10b due to changes in the (C) sodium and carboxylate binding site.

Examining the substrate and sodium binding sites, it is immediately apparent that the structural changes observed between Ci-Na+ and Ci-Na+-PF2 is triggered by NaCT-substrate/inhibitor interactions (Figure 3C). In particular, PF2 makes hydrogen bonds with N141, T142, T227, G228, N465 and T508, bridging the core of the transport domain and HPin and TM10b. Consequently, release of carboxylate substrate relieves this structural restraint to trigger the structural movements of HPin and TM10b that are subsequently propagated to the interfacial helices through the hinge loop connecting HPina to H6a. Notably, such differences between the Ci-Na+ and Ci-Na+-PF2 structures are inconsistent with the predictions made by the sequential binding, conformation selection coupling mechanism 35.

2.3. Binding site flexibility upon substrate release

While the cryo-EM maps were sufficient to build atomic models for the entire NaCT transporter in all states, markedly weaker density was observed near Na2 in the Ci-Na+ and Ci-apo states (Figure 2). Furthermore, we recognized that, while traditional model refinement provides a single protein model for each experimental condition, the varying strength of the experimental density might reflect differences in mobility across the protein structure. We therefore next examined changes in NaCT flexibility between the three conditions by directly comparing the cryo-EM densities, along with the convergence and atomic mobility in the refined models.

Examining the equivalently scaled Coulombic potential maps, we immediately recognized that the density for TM10b of NaCT was poorly resolved for the Ci-Na+ and Ci-apo states, relative to Ci-Na+-PF2 (Figures 4A-C), even though the densities for the rest of the protein were well defined. This suggested greater structural flexibility of TM10b near Na2 in the absence of substrate; as alternative mechanisms for the observed map differences, such as radiation damage or poor particle alignment, would not result in region-specific effects. Furthermore, density at NaCT’s sodium binding sites or TM10b could not be recovered when reprocessing the particle stack with C1 symmetry, or after symmetry expansion and 3D variability analysis (Video S1). This indicates that the absence of density in this region is not due to a subpopulation of NaCT in a discrete alternative conformation. While the cryo-EM maps alone are insufficient to resolve if sodium is binding to NaCT, the cation alone is clearly insufficient to drive a structural rigidification of the substrate binding site in the protein. Ultimately, this similarity between the Ci-Na+ and Ci-apo structures, and differences between the Ci-Na+ and Ci-Na+-PF2 structures, are again in conflict with the predictions of sequential binding via a conformational selection mechanism 35.

Figure 4. Transport domain flexibility changes between NaCT’s Ci-Na+-PF2, Ci-Na+, and Ci-apo states.

Figure 4.

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Equivalently scaled unsharpened cryo-EM maps of the (A) Ci-Na+-PF2, (B) Ci-Na+, and (C) Ci-apo states, with refined atomic models shown in blue, green, and pink, respectively. Structures of the (D) Ci-Na+-PF2, (E) Ci-Na+, and (F) Ci-apo states, colored by the normalized model B-factors. NMR-style analysis of the (G) Ci-Na+-PF2, (H) Ci-Na+, and (I) Ci-apo states from 10 independent refinements.

Having identified this region of state-dependent flexibility in NaCT, we next sought to capture these structural dynamics in the states’ corresponding atomic models. Although the refined models of NaCT across states were generally similar, atomic B-factors were elevated near the Na2 site of the Ci-Na+ and Ci-apo models (Figures 4D-F). Similarly, Q-scores 58 for TM10b were notably reduced in the Ci-Na+ and Ci-apo models, providing quantitative validation that this region is less well resolved (Figure S4). However, we suspected a single atomic model might not adequately capture the structural ensemble of NaCT in each experimental map and the corresponding state. Therefore, we conducted an NMR-style analysis using multiple independent rounds of simulated annealing and model refinement 35. In this method, the rigid parts of the protein will converge to the same coordinates, while the mobile regions will be at distinct atomic positions that fit equally well into the map after each run. This method provides a visualization of mobile regions in protein structures refined against cryo-EM maps, though it cannot describe the true atomic coordinates of the protein's flexible regions due to the lack of experimental density. As expected, the models converged across most of the well-resolved regions of NaCT protein and its transport domain (Figures 4G-I). However, significant structural heterogeneity was observed near the Na2 site and in TM10b in the absence of PF2.

3. Discussion

VcINDY’s conformational selection model provided the first mechanistic model for energy-coupling by a DASS family member 35. However, significant sequence and functional differences between the prokaryotic VcINDY and human NaCT suggested the human protein might not use an identical mechanism to couple cation and carboxylate binding. Here, we determined structures of NaCT in its Ci-Na+-PF2, Ci-Na+ and Ci-apo states to understand this eukaryotic protein’s coordination of sodium and substrate-mimicking inhibitor, PF2.

In the absence of sodium, both NaCT and VcINDY show greater flexibility in the Na2 site and TM10b helix than the Na1 site. Remarkably, the PF2/sodium-induced structural changes of NaCT differ significantly from those observed in its bacterial homolog VcINDY. First, NaCT is rigid at the Na1 site in the absence of sodium, whereas the structure of VcINDY is highly flexible in the absence of a cation. Second, the presence of sodium was sufficient to fix the loop positions at the Na2 site in VcINDY for dicarboxylate binding, whereas this site and TM10b in NaCT become rigid only in the presence of both sodium and PF2. In summary, for VcINDY the binding site structures are similar between Ci-Na+-S and Ci-Na+ and different between Ci-Na+ and Ci-apo, indicating that sodium ions alone can select a conformation for dicarboxylate binding. In contrast, NaCT’s Ci-Na+ and Ci-apo share similar binding site structures, which are different from those of Ci-Na+-PF2. This shows that sodium alone is insufficient to select a configuration in NaCT for substrate binding. These contradictions demonstrate that NaCT couples sodium and dicarboxylate binding by a mechanism different from VcINDY’s sodium-induced conformational selection.

The origin of NaCT and VcINDY’s distinct sodium and substrate coupling mechanisms are unclear due to differences in sequence and function between the proteins. The proteins are distant homologs, with ~30% pairwise amino acid sequence identity. In addition, NaCT binds 4 sodium ions 10,50, versus VcINDY’s 3 co-transported cations 51, which may be important to the coupling mechanism. Furthermore, while the sodium binding sites Na1 and Na2 have been identified and are structurally homologous between both proteins, the remaining cation sites remain unknown. Frustratingly, our high-resolution structures of NaCT in saturating sodium conditions do not give any indication of the locations of these additional ions.

Finally, the sequential binding and co-transport of sodium and carboxylates by DASS proteins are typically studied through their import reactions. Under these conditions, sodium and substrate binding occur when the protein is in an outward-facing state and in the presence of a 10-fold transmembrane sodium gradient. In contrast, our results describe the cytoplasmic release of carboxylate and sodium. The pseudosymmetry of the DASS family 25,36, along with the transporters’ reversibility 22, suggests the inward-facing and outward-facing reactions are structurally symmetric, with TM10b rigidly packed against the scaffold domain and TM5b mobile prior to loading of sodium or substrate. However, structural changes in the presence of sodium or substrate to enforce coupled transport will depend on the unique packing interactions for each conformation in each protein of the DASS family 31. Therefore, fully exploring sodium and substrate-coupled transport in the DASS proteins will require capturing a complete reaction cycle, including outward-facing states, for both prototype VcINDY and the human NaCT.

Tentatively, the structural differences between NaCT’s Ci-Na+-PF2, Ci-Na+, and Ci-apo states suggest one possible mechanism for substrate and sodium coupling. NaCT’s carboxylate coordinating residues are directly linked to the Na1 and Na2 binding sites. Consequently, the human protein’s cation binding may be substrate dependent, with the occluded Na1 and Na2 sites in our Ci-Na+-PF2 structure suggesting the sodium and carboxylate binding is simultaneous. This effectively maintains the transporter in an apo state unless both sodium and carboxylate substrate are present, and thereby prevents non-productive leakage where only the cation is transported across the membrane. Such a coupling mechanism satisfies the charge compensation requirement for translocating highly-charged substrates across the membrane.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Da-Neng Wang (da-neng.wang@med.nyu.edu).

Materials availability

All unique reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

  • Cryo-EM maps and models have been deposited to Protein DataBank, and are publicly accessible. NaCT-Na+-PF2 structure map and model have the following accession codes: 30UT (PDB), EMD-58071 (EMDB). NaCT-Na+ structure map and model have the following accession codes: 30UU (PDB), EMD-58072 (EMDB). NaCT-apo structure map and model have the following accession codes: 30UV (PDB). EMD-58073 (EMDB).

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported is available from the lead contact upon request.

STAR*Methods

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

T. ni BTI-Tn-5B1–4 cells and Sf9 cells were used for the overexpression of the human NaCT protein.

METHOD DETAILS

Overexpression and purification

NaCT was expressed and purified as previous described with minor modifications 29. Briefly, T. ni BTI-Tn-5B1–4 cells were transduced with baculovirus bearing the wild-type SLC13A5 gene with an N-terminal decahistidine-TEV tag. Cells were lysed in a buffer of 50 mM Tris pH 8.0, 400 mM NaCl, 10 mM lithium citrate and 10% glycerol. Membranes were collected by ultracentrifugation and solubilized in a buffer of 50 mM Tris pH 8.0, 200 mM NaCl, 10 mM lithium citrate, 10% glycerol and 1.3% DDM. NaCT was subsequently purified on a Ni2+-NTA affinity column in buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM Li-Citrate, 10% glycerol, 500 mM imidazole, 0.1% DDM and 0.5 mM TCEP. Amphipol PMAL-C8 (Anatrace) was added to the sample at a ratio of 5:1 amphipol/protein (w/w), and the DDM detergent was removed with Bio-Beads SM2 (Bio-Rad) absorption. The sample was further purified by size-exclusion chromatography in buffer containing 25 mM Tris pH 7.5, 0.1 mM TCEP and either 300 mM NaCl and 100 μM PF2 (for NaCT-Na+-PF2) or 300 mM NaCl (for NaCT-Na+) or 100 mM choline chloride (NaCT-apo).

Cryo-EM specimen preparation and image processing

All cryo-EM grids were prepared by applying 3 μl of protein at around 0.75 mg ml−1 to a glow-discharged UltrAuFoil R1.2/1.3 300-mesh grid (Quantifoil) and blotted for 2.5–4 s under 100% humidity at 4°C before plunging into liquid ethane using a Mark IV Vitrobot (FEI).

Micrographs of the NaCT–Na+-PF2 complex were acquired on a Titan Krios microscope with a K3 direct electron detector and an energy filter slit width of 20 eV. The magnification used was 105,000×, with a physical pixel size of 0.826 Å. Leginon 59 was used to for ice thickness targeting, measured as described previously 60, resulting in 1,585 and 2,464 micrographs being collected from 0° and 40° tilted specimens, respectively. Each micrograph was dose-fractioned over 50 frames, with an accumulated dose of 63 e− Å−2. Frame alignment was performed with Relion 4 61. Aligned and dose weighted micrographs were imported to Warp 62. After particle picking and CTF estimation in WARP, micrographs with an overall resolution worse than 12 Å were excluded from subsequent steps. Initial Warp particle picks were cleaned up in CryoSparc 63 with two-dimensional and three-dimensional classification, and then used for repicking using Topaz 64 to yield 2,620,564 particles from all micrographs. In CryoSparc, these particles were subjected to multiple rounds of three-dimensional classification to produce a set of 1,000,432 particles, which generated a 3.11 Å reconstruction in nonuniform refinement with C2 symmetry imposed. This map and particle set were then used for Relion 65 particle polishing. The particles were re-imported in CryoSparc for global and local CTF optimization and subsequent nonuniform refinement to yield a final 2.53 Å reconstruction with C2 symmetry imposed.

Micrographs of the NaCT-Na+ state were acquired on a Titan Krios microscope with a K3 direct electron detector and an energy filter slit width of 20 eV. The magnification used was 105,000×, with a super-resolution pixel size of 0.426 Å. Leginon was used to target holes with 10 to 70 nm of ice, measured as described previously, resulting in 2,353 and 3,192 micrographs being collected from 0° and 40° tilted specimens, respectively. Each micrograph was dose-fractioned over 48 frames, with an accumulated dose of 53.6 e− Å−2. Frame alignment was performed with MotionCorr2 66 under control of Appion. Aligned, dose-weighted micrographs were imported to Warp. After particle picking and CTF estimation in Warp, micrographs with an overall resolution worse than 8 Å were excluded from subsequent steps, with the initial set comprising 2,349,354 particles. Warp-picked particle sets from the tilted and untilted micrographs were cleaned using CryoSparc’s two-dimensional and three-dimensional classification, and then combined. These 1,421,195 particles were subjected to multiple rounds of three-dimensional classification to produce a set of 951,970 particles, which generated a 2.63 Å reconstruction in nonuniform refinement with C2 symmetry imposed. This map and particle set were then used for Relion particle polishing. The particles were re-imported in CryoSparc for subsequent nonuniform refinement to yield reconstructions of 2.56 Å and 2.51 Å with C1 and C2 symmetry imposed, respectively. The C2 map and particles were then used for symmetry expansion and 3D variability analysis with a mask around the NaCT protein.

Micrographs of the NaCT-apo state were acquired on a Titan Krios microscope with a K3 direct electron detector and an energy filter slit width of 20 eV. The magnification used was 105,000×, with a super-resolution pixel size of 0.426 Å. Leginon was used to target holes with 15 to 95 nm of ice, measured as described previously, resulting in 2,237 and 5,473 micrographs being collected from 0° and 40° tilted specimens, respectively. Each micrograph was dose-fractioned over 48 frames, with an accumulated dose of 58.0 e− Å−2. Frame alignment was performed with MotionCorr2 66 under control of Appion, and aligned. Dose-weighted micrographs were imported to Warp. After particle picking and CTF estimation in Warp, micrographs with an overall resolution worse than 8 Å were excluded from subsequent steps. Initial Warp picks of 1,117,117 particles were cleaned up using CryoSparc’s two-dimensional and three-dimensional classification, and then used for repicking using Topaz to yield 3,170,073 particles from all micrographs. In CryoSparc, these particles were subjected to multiple rounds of three-dimensional classification to produce a set of 1,503,602 particles, which generated a 2.73 Å reconstruction in nonuniform refinement with C2 symmetry imposed. This map and particle set were then used for Relion particle polishing. The particles were re-imported in CryoSparc for subsequent nonuniform refinement to yield a final 2.49 Å reconstruction with C2 symmetry imposed.

Atomic models were built in Coot 67 and refined with Phenix.real_space_refine 68.

The NMR-style analysis was performed as previously described 35, using 5 independent runs of phenix.real_space_refine to refine the models, with ions and ligands removed, against the unsharpened maps with a unique computational seed for each run. Each refinement was performed with simulated annealing, without NCS constraints or secondary structure restraints, and a refinement resolution limit of 2.53 Å for all maps and models. When comparing the local density, unsharpened maps were scaled to equivalent contours using the scaffold domain’s volume as an internal standard after extracting with phenix.map_box 68. Figures were made using UCSF Chimera 69, ChimeraX 70, and PyMOL 71.

METHOD DETAILS; QUANTIFICATION AND STATISTICAL ANALYSIS

Cryo-EM data collection and refinement statistics are summarized in Table S1.

Supplementary Material

2

Video S1. 3D variability analysis of the NaCT-Na+ map, related to Figure 4. No sodium density is seen at Na1 or Na2 site in the analysis, indicating that the absence of density in this region is not due to a subpopulation of the transporter protein in a discrete alternative conformation

Download video file (3.8MB, mp4)
1

Supplemental information

Supplemental methods, Figures S1–S6 and Video S1

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, peptides, and recombinant proteins
Choline Chloride Sigma Cat#C1879
ESF 921 Insect Cell Culture Medium Expression systems Cat#96–001-01
Lithium Citrate Sigma Cat#213209
Lipofectamine 3000 Invitrogen Cat#L3000001
PF-06649298 (PF2) Sigma Cat#AABH9A95CB25
n-Dodecyl-β-D-maltopyranoside (DDM) Anatrace Cat#D310
PMAL-C8 Anatrace Cat#P5008
Bio-Beads SM-2 adsorbants BioRad Cat#1523920
Ni2+-NTA Resin Qiagen Cat#30210
Deposited data
Human NaCT-Na+-PF2 This study PDB: 30UT EMD-58071
Human NaCT-Na+ This study PDB: 30UU EMD-58072
Human NaCT-apo This study PDB: 30UV EMD-58073
Experimental models: cell lines
Tni Cells Expression systems Cat#94–002F
Sf9 Cells Expression systems Cat#94–001F
Recombinant DNA
Deca-histidine-TEV-pFastBac- NaCT Sauer et al. 202129 N/A
Software and algorithms
RELION4.0 Zivanov et al. 202261 http://www2.mrc-lmb.cam.ac.uk/relion/
CryoSparc v3.3.1 Punjani et al. 201763 https://cryosparc.com/
Leginon v.3.6 Cheng et al. 202159 https://leginonorg.github.io/leginon/leginon/
COOT Emsley et al. 200467 http://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot
Phenix.real_space_refine Liebschner et al. 201968 https://www.phenix-online.org/
UCSF Chimera Pettersen et al. 200469 http://www.cgl.ucsf.edu/chimera
UCSF ChimeraX Meng et al. 202370 https://www.cgl.ucsf.edu/chimera x/
WARP Tegunov et al. 201962 https://warpem.github.io/warp/home/warp/
Topaz Bepler et al. 201964 https://cb.csail.mit.edu/cb/topaz/
PyMOL DeLano71 https://pymol.org/2
Prism Graphpad http://www.graphpad.com/
Other
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Highlights.

  • In the structure with both Na+ and substrate the Na+ and substrate sites are rigid

  • In Na+ or in apo the Na+ and substrate sites are found to be flexible

  • These suggest a simultaneous binding mechanism for Na+-substrate coupling

Acknowledgements

This work was financially supported by the NIH (R01NS108151, R01DK135088 and R01GM121994), the G. Harold and Leila Y. Mathers Foundation, and the TESS Research Foundation. D.B.S. was supported by the American Cancer Society Postdoctoral Fellowship (129844-PF-17–135-01-TBE) and the Department of Defense Horizon Award (W81XWH-16–1-0153). The cryo-EM data was collected at the NYU Cryo-Electron Microscopy Core Facility (RRID: SCR_019202), which is partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI P30CA016087, and at the Simons Electron Microscopy Center at the New York Structural Biology Center, which is supported by the Simons Foundation (SF349247). We are grateful to E. Eng for technical support. EM data processing used computing resources at the HPC Facility of NYULMC.

Footnotes

Declaration of interests

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2

Video S1. 3D variability analysis of the NaCT-Na+ map, related to Figure 4. No sodium density is seen at Na1 or Na2 site in the analysis, indicating that the absence of density in this region is not due to a subpopulation of the transporter protein in a discrete alternative conformation

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1
NIHMS2204263-supplement-1.pdf (25MB, pdf)

Data Availability Statement

  • Cryo-EM maps and models have been deposited to Protein DataBank, and are publicly accessible. NaCT-Na+-PF2 structure map and model have the following accession codes: 30UT (PDB), EMD-58071 (EMDB). NaCT-Na+ structure map and model have the following accession codes: 30UU (PDB), EMD-58072 (EMDB). NaCT-apo structure map and model have the following accession codes: 30UV (PDB). EMD-58073 (EMDB).

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported is available from the lead contact upon request.

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