Lee CH , Lü W , Michel JC , Goehring A , Du J , Song X , Gouaux E .

Howard Hughes Medical Institute , R37 NS038631 NINDS NIH HHS

	Extended Data Figure 2. Electrophysiology and Western analysis of GluN1 δ/GluN2B δ receptor combinationsa, b, c, Representative TEVC currents recorded for oocytes expressing GluN1 δ4 and (a) GluN2B δ1 or (b, c) GluN2B δ3 receptors in response to agonist (100µM glycine and 100 µM glutamate, bars, 20 sec) or agonist plus 1 mM MgCl2 (indicated) after soaking oocytes in the (a, b) absence or (c) presence of 5mM DTT. d, Western blot analysis of oocytes demonstrating spontaneously crosslinking cysteines (Lys216Cys) introduced at the GluN2B δ3 intersubunit interface. Oocytes were soaked in the absence (left lanes) or presence of 5mM DTT (right lanes) before processing for Western analysis using an anti-GluN2B antibody. Filled and open triangles indicate positions of crosslinked and monomeric GluN2B, respectively. e, Graph of mean agonist-induced inward currents from four reduced oocytes expressing GluN1 δ4 and GluN2B δ3 in the absence (G/G, −25 ± −4 nA) or presence of 1mM MgCl2 (G/G/Mg2+, 8 ± 5 nA). Error bars represent s.e.m. The p value is <0.001 for the paired T-test (asterisk). f, Representative TEVC currents recorded in response to agonist (100µM glycine and 100 µM glutamate bars, 10 sec) or agonist plus 1 mM MgCl2 for oocytes expressing constructs similar to the GluN1 δ2/GluN2B δ2 receptor combination with the following exceptions: GluN1 subunit, Asp656 (wt), Gly636Arg and Lys741Asp; and GluN2B subunit, Glu654 (wt), Glu655 (wt), and Lys216 (wt). g, Binding constants for the GluN1 δ2/GluN2B δ2 construct.
	Extended Data Figure 3. 2Fo-Fc electron density maps of the GluN1/GluN2B NMDA structurea, The electron densities associated with the GluN1 ATD (chain A) contoured at 1.7 σ, (b) the GluN1 LBD (chain A) contoured at 1.6 σ, (c) the TMD of the entire tetrameric receptor contoured at 1.0 σ and (d) the TMD of a single GluN2B subunit (chain D), showing the pore loop, also contoured at 1.0 σ. Electron density maps and structures were derived from Data set 1/Structure 1 for panels (a) and (b) and from Data set 2/Structure 2 for panels (c) and (d) (see Extended Data Table 2).
	Extended Data Figure 4. Analysis of spontaneous crosslinking of single cysteine point mutants introduced in the GluN2B ATD of the GluN1/GluN2B receptor complexa, Western blot analysis of single cysteine mutants in the α5 helix of the GluN2B subunit. Solubilized extracts of HEK293S GnTI cells expressing a C-terminal GFP-StrepII tag GluN2B construct (GluN2B δ1) containing mutants as indicated with untagged GluN1 (GluN1 δ1) were analyzed by Western blot using an anti-GFP polyclonal antibody. The open and filled arrows correspond to monomeric and dimeric GluN2B bands, respectively. b, Coomassie stained SDS-PAGE analysis of spontaneous crosslinking of GluN2B K216C containing receptor. Left and right lanes illustrate samples with different concentrations of protein for GluN1/GluN2B and GluN1/GluN2B K216C receptors. The asterisk indicates GluN1 monomer while the open and filled arrows correspond to monomeric and dimeric GluN2B bands, respectively.
	Extended Data Figure 5. Structural analyses and electron density maps of GluN1/GluN2B ATD heterodimer in the full-length NMDA structurea, Intersubunit distance between the indicated marker atoms and angle of domain closure in the soluble ATD structure (PDB 3QEM, left panel) or full-length ATD structure (right panel). b, Superposition of the full-length GluN1 (blue)/GluN2B (orange) ATD heterodimer onto the soluble heterodimer structure (PDB 3QEM, light grey) by aligning the indicated helices (green) in the R1 lobe of GluN2B. c, Fo-Fc omit electron density map for Ro25-6981 bound at the GluN1/GluN2B ATD heterodimer interface (chains A and B), contoured at 3σ (Data set 1/Structure 1). d, Anomalous difference electron density of Tb3+ (blue mesh) near the R1-R2 hinge of a single GluN2B ATD (chain B, Data set 3), contoured at 3.5 σ. e, Superposition of the LBD layer of the low resolution GluN1/GluN2B receptor (light blue, Data set 4/Structure 4) onto the LBD layer of the high resolution K216C receptor (magenta, Data set 1/Structure 1) illustrates the relative difference in ATD conformations between the two receptor structures (see Extended Data Table 2). Shown is the most 'open' conformation of the ATDs derived from one of the two independent receptors in the asymmetric unit of Data set 4/Structure 4.
	Extended Data Figure 6. LBD ligand electron densities and conformationsFo-Fc omit electron density maps for (a) ACPC bound to GluN1 LBD (chain A) and (b) t-ACBD bound to GluN2B LBD (chain D), contoured at 3 σ and 2.5 σ, respectively (Data set 1/Structure 1). c, d, e, Comparison of LBD in the full-length GluN1/GluN2B structure to isolated structures by aligning the D1 lobe. The angle of rotation relative to beta strand 10 is indicated for each. c, The ACPC-bound GluN1 LBD of the full-length structure (chain A, blue) is more open than the ACPC-bound isolated GluN1 LBD structure (PDB 1Y20, grey). d, The ACPC-bound GluN1 LBD of the full-length structure (chain A, blue) is more open than the glycine-bound isolated GluN1 LBD structure (PDB 2A5T, chain A, grey). e, The t-ACBD-bound GluN2B LBD of the full-length structure (chain D, orange) has a similar domain closure to the glutamate-bound isolated GluN2B LBD (PDB 2A5T, chain B, grey). f, GluN1/GluN2B LBD heterodimer (chains A and D) from the full-length receptor structure showing the separation of the D2 lobes, measured using the α-carbon atoms of residues Gly 664 and Gly 662, respectively. g, A similar measurement as in (f) using the equivalent residues in the context of the rat glycine/glutamate bound isolated GluN1/GluN2A LBDs (PDB 2A5T). h, The same measurement as in (g), except in the GluN1 antagonist/Glu2A glutamate-bound conformation (PDB 4NF4). Structures shown in panels c-f were derived from Data set 1/Structure 1 and are similar in conformation to the related domains derived from Data set 2/Structure 2 (see Extended Data Table 2).
	Extended Data Figure 7. Structural analyses of the transmembrane domain of NMDA receptora, Alpha-carbon superposition of the M3 helices of the GluN1/GluN2B NMDA receptor (Data set 2/Structure 2) onto the corresponding M3 regions of GluA2 receptor (PDB 3KG2; grey). Rmsd is 1.89 à for 144 aligned α-carbon atoms. The GluN1 subunits are blue and the GluN2B subunits are yellow. b, Amino acid sequence alignment of the NMDA receptor and the KcsA channel in the M2 and M3 regions using Promals3D (http://prodata.swmed.edu/promals3d/promals3d.php). c, Superposition of the four M2 helices of the NMDA receptor onto the corresponding four M2 regions of the KcsA channel (PDB 1K4C; residues 61–75). Rmsd is 1.86 à . Only chains B and D of the NMDA GluN2B subunits are shown. d, Residual electron density in the central vestibule. Fo-Fc electron density in the central vestibule is shown for the GluN1/GluN2B receptor from Data set 2/Structure 2. For clarity, chain C is removed. e, Fo-Fc electron density map in the central vestibule derived from Data set 1/Structure 1. For clarity, chain B is removed. f, The same electron density map as shown in panel (e) except that the structure has been rotated by ~90° around the pore axis and chain C of the GluN1 subunit has been removed for clarity. All maps are contoured at 2.8 σ.
	Extended Data Figure 8. Comparison of LBD layers and LBD-TMD linkers between the NMDA receptor and the GluA2 receptor structuresa, View from the extracellular side of the membrane of the connections between the TMD and LBD domains of the GluN1/GluN2B structure and of the GluA2 structure (PDB 3KG2), showing the relative rotation of GluA2 layer by ~35°. The S2 segment resides within the LBD. The LBD-M3 linkers are highlighted. b, The LBD-M1 linkers are highlighted. c, The LBD-M4 linkers are highlighted. Shown in all panels are structures derived from Data set 1/Structure 1.
	Figure 1. Architecture, symmetry and domain organization of the GluN1/GluN2B NMDA receptora, View of the receptor complex, parallel to the membrane, with the GluN1 subunits in blue and the GluN2B subunits in orange. The ligands Ro25-6981, ACPC and t-ACBD are in space filling representation. b, View of complex rotated ~120° around overall the 2-fold axis of the receptor. The approximate position of the overall 2-fold axis is shown by a vertical gray bar in the center of the ATD layer. Structure 2 is shown.
	Figure 2. ATD arrangement, cation binding sites and conformational mobilitya, View of the ATD layer along the overall 2-fold axis, from the extracellular side of the membrane, centered on the overall 2-fold axis, and showing the relative location of the underlying LBD layer. Ro25-6981 is green and the K216C disulfide is yellow. The arrangements of subunits for ATD and LBD layers are shown as insets. b, The inverted ATD heterodimeric ‘V’ straddles GluN1 and GluN2B LBD subunits on different local LBD heterodimers. Whereas the ATD R2 lobes interact with the LBDs, the R1 lobes cradle bound Ro25-6981 at an ATD subunit interface. Structure 1 is shown in panels (a) and (b) c, Tb3+ binding sites. Shown is an anomalous difference electron density map, contoured at 3.5 σ (pink mesh). Sites Tb1 and Tb2 are located at the ‘hinge’ between the R1 and R2 lobes whereas sites Tb3 and Tb4 are at receptor-receptor contacts in the crystal lattice. d, Shown are the ATD and LBD extracellular domains derived from the two low resolution GluN1/GluN2B receptor structures (Extended Data Table 2; Data set 4/Structure 4) where the GluN2B subunits do not harbor the K216C disulfide bridge, illustrating the conformational mobility of the ATD layer. The angles between the α5 helices of the GluN2B subunits for each of the two independent receptor complexes in the asymmetric unit illustrate the conformational mobility of the ATD layers.
	Figure 3. LBD layer forms a ring-like structurea, The GluN1/GluN2B LBD and TMD, showing that the pseudo 2-fold axes of the B/C and A/D LBD heterodimers diverge with an angle of 60°. The boxed areas define regions of LBD dimer-dimer contacts shown in panels (g) and (h). b, View of the antagonist-bound state of the GluA2 AMPA receptor, which shows that the 2-fold axes of the LBD dimers diverge by an angle of 40.9°c, View from the extracellular side of the membrane, along the overall 2-fold axis of the receptor, showing the LBDs of the GluN1 and GluN2B subunits, with the LBD heterodimer interface of the B/C subunits emphasized by a box. d, GluA2 LBD layer, illustrating how the interface between the B/C and A/D subunits has increased in comparison to the NMDA receptor LBD layer. e, Schematic of the LBD layer, showing the NMDA receptor B/C and A/D heterodimers as rectangles (solid lines) and illustrating the translational shift of the A/D subunits in the AMPA receptor (dotted lines). The asterisk indicates the dimer-dimer interface. f,g,h, Closeup view of the canonical D1-D1 intradimer interface32, together with views of the interactions at the interdimer interfaces in panels (g) and (h). The domains from Structure 2 are shown, with GluN1 subunits in blue and GluN2B subunits in orange.
	Figure 4. The ATDs participate in extensive contacts with the LBD layera. Surface representation of the ATD and LBD domains, illustrating how the R2 lobe of the GluN1 subunit is poised above its cognate GluN1 LBD and also near the D1-D1 LBD dimer interface and in (b) how the R2 lobe of the GluN2B subunit participates in contacts with its cognate GluN2B LBD, near an inter LBD dimer interface. c. Close up views of potential interactions between the GluN1 R2 lobe and the GluN1 LBD and (d) between the GluN2B R2 lobe with regions on its GluN2B LBD. Note that the GluN2B R2 lobe is also near helices G and F and loop 2 of the GluN1 LBD. In (a) and (b) the black dots define the approximate intra- and interdimer LBD interfaces, respectively. Structure 1 is shown in all panels.
	Figure 5. Transmembrane domain architecture, symmetry and coupling to LBDa, View of the TMD parallel to the membrane. GluN1 subunits are blue and the GluN2B subunits are orange. b, View of the TMD, along the pore axis, from the cytoplasmic side of the membrane. c, View of a solvent accessible surface carved along the pore axis using the computer program HOLE, parallel to the membrane, showing that the M3 bundle crossing near the extracellular side of the membrane and the entry into the selectivity filter region, from the central aqueous vestibule, form constrictions in the pore. The color coding for the dots that indicate the pore radius is 1.15 à < green < 2.3 à < blue. Because a number of side chains are not included in the structure, due to the moderate resolution of the diffraction data, the size of the pore is approximate. d, View of the extracellular ends of the M3 helices of the NMDA receptor. We have highlighted as spheres the α-carbon atoms for residues Thr 646 and Ala 645 in the GluN1/GluN2B structure, respectively. The distances between neighboring atoms are 6.2, 8.0, 5.4 and 7.1 à , starting from the α-carbon of GluN2B on the left and going clockwise. e, View of the intracellular ends of the TMD of the NMDA receptor in comparison with KcsA. Here, the M2 helices of the NMDA receptor were superimposed on the corresponding helices in KcsA, showing the deviation from 4-fold symmetry. f, Side view of the TMD showing a positive electron density feature (green mesh) in the central vestibule, calculated using Fo-Fc coefficients and phases from the refined structure. The map is contoured at 2.8 σ. Data set 2 and Structure 2 were employed in all panels (Extended Data Table 2).
	Figure 6. Schematic of the NMDA receptora, Shown is a single ATD heterodimer, two LBD ‘clamshells’ residing in different LBD heterodimers, and the TMD of GluN2B subunits, emphasizing only the M2, pore loop and M3 elements. The line connecting the M3 helix on the right is ‘broken’ to illustrate that it is connected to the GluN2B LBD ‘behind’ the shown GluN1 LBD. Double-headed arrows suggest possible movements of ATDs within an ATD heterodimer. b, Rotation of the receptor schematic shown in panel (a) by ~120° showing two ATD heterodimers, a single LBD heterodimer and the TMD of GluN1 subunits. Double-headed arrows show conformational movements between ATD heterodimers observed in the structures described here. The α5 helices, harboring the K216C crosslink, are shown as rectangles at the R2-R2 interface. In both schematics, we emphasize how the R2 lobes of the ATDs are positioned such they could modulate inter- and intradimer LBD interfaces and, in turn, the ion channel gate.

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