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Fig. 1. The structure of xlσ1Rclosed-endo compared to hσ1RPD144418.a The structure of the hσ1RPD144418 (PDB: 5HK1) homotrimer is viewed parallel to the membrane, with two protomers rendered in the surface mode and one in the cartoon. The helices are rendered as cylinders, and are labeled as α1–α5 from the amino- (N-) to the carboxy- (C-) terminus throughout the paper. The cupin-fold domain is rendered as a blue β barrel, with the red sheets indicating the hydrogen bonds to disrupt for ligand entry as suggested previously20. The C-terminal two-helix bundle (α4/α5) is colored in green, while the N-terminal part is orange. The relative position of the ER membrane is indicated by two gray lines. b The close-up view of one protomer from panel (a). The bound ligand, PD144418, is displayed as magenta sticks. The two pathways proposed for ligand entry are indicated by two magenta arrows, PATH1 and PATH2. c Left, superposition of the xlσ1Rclosed-endo trimer (in green) and the hσ1RPD144418 trimer (in brown) viewed perpendicular to the membrane from the cupin-fold side. Right, superposition of one xlσ1Rclosed-endo protomer and one hσ1RPD144418 protomer viewed parallel to the membrane. d One xlσ1Rclosed-endo protomer is rendered in both cartoon and surface modes in a “slab” view, in which the internal cavity is displayed as a gray shadowy compartment within the β barrel. The purple mesh shows the simulated annealing Fo–Fc map contoured at 3.0 σ level corresponding to an unidentifiable molecule.
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Fig. 2. Soaking PRE084 into the closed xlσ1R structure.a Crystal packing of two adjacent xlσ1Rclosed-endo trimers. Protomers A (in green) and C (in orange) are from the bottom trimer, while Protomer D (in yellow) is from the top trimer, and the other two protomers of the top trimer are not displayed for a clearer view. The tip region of the cupin-fold domain (Trp133–Tyr144) of Protomer C is rendered in spheres. b The close-up view of Protomers A (in green), C (in orange), and D (in yellow) from panel (a). The residues participating in the close contacts are labeled and are displayed in spheres. c Left, one xlσ1Rclosed-PRE084 trimer in the cartoon mode viewed perpendicular to the membrane from the cupin-fold side. Right, the close-up view of the same trimer is viewed parallel to the membrane. The ligand PRE084 is displayed in sticks, and the purple mesh shows the simulated annealing Fo–Fc omit map contoured at 3.0σ level corresponding to PRE084.
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Fig. 3. An open-like conformation of xlσ1R.a Superposition of the xlσ1Ropen-endo trimer (in green) and the xlσ1Rclosed-endo trimer (in orange), viewed parallel to the membrane. b Superposition of one protomer of xlσ1Ropen-endo (in green), one protomer of xlσ1Rclosed-endo (in orange), and each protomer (in red, cyan, and blue) of the hσ1RPD144418 trimer, viewed parallel to the membrane. c Left, the close-up view of the panel (a), showing the difference in α4/α5 between the two structures. Leu179 and Tyr203 are rendered in sticks. Right, the electron density map for α4/α5 of the xlσ1Ropen-endo structure, viewed perpendicular to the membrane from the cupin-fold side. Tyr203 is labeled, and the blue mesh shows the simulated annealing 2Fo–Fc map contoured at 1.2σ level. d A solvent pathway connecting the ligand-binding site of xlσ1Ropen-endo and the outside milieu rendered in a pink surface. Left, viewed parallel to the membrane. Right, viewed perpendicular to the membrane from the membrane side. The approximate dimension of the entrance is indicated by a dashed cross. e, f Protomer F (in green) and D (in yellow) of xlσ1Ropen-PRE084-co, and Protomer B (in orange) of xlσ1Ropen-PRE084-soak are displayed side-by-side to compare the rotamer of Tyr203. The blue mesh shows the simulated annealing 2Fo–Fc map contoured at 1.2σ level, and the purple mesh shows the simulated annealing Fo–Fc omit map contoured at 3.0 σ level corresponding to PRE084 (in cyan sticks).
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Fig. 4. Blocking the putative entrance in PATH2.a One protomer of xlσ1Ropen-endo viewed perpendicular to the membrane from the membrane side. Left, the residues surrounding the entrance are displayed in sticks. Right, the surface representation shows an “egg” shape of the entrance residues in three parts: the rounded end (in blue), the middle (in yellow), and the pointed end (in red). b, c The stoichiometry (available binding site number per protomer, panel b) and the equilibrium dissociation constant (Kd, panel c) determined by ITC for the xlσ1R-C179/C203 protein in the indicated conditions. All ITC measurements were repeated with biologically independent samples. “Before oxidation”: n = 5. “After oxidation”: n = 3. “Re-reduction”: n = 4. Two-tailed Student’s t-test was performed between the conditions of “Before oxidation” and “After oxidation”, and between the conditions of “After oxidation” and “Re-reduction”. The p values are provided in a Source Data file. **p < 0.01. d Modification of mPEG-Mal-5K to the xlσ1R-C179/C203 protein in the indicated conditions analyzed by SDS-PAGE and Coomassie blue staining. The wild-type (WT) xlσ1R served as the control. The pound sign (#) indicates that the protein contains the extra C91S mutation (see “Methods” for details). The black arrowheads labeled with 0, +1, and +2 indicate the band positions without, with one, and with two mPEG-Mal-5K modification(s). The red arrowhead indicates a minor impurity band near 50 kDa, which does not affect the gel analysis. The modification experiment was repeated three times independently with similar results. e, f The stoichiometry (available binding site number per protomer, panel e) and the equilibrium dissociation constant (Kd, panel f) determined by ITC for the indicated xlσ1R proteins before and after the mPEG-Mal-5K modification. All ITC measurements were repeated with biologically independent samples. “Before modification”: n = 4 for xlσ1R WT, n = 4 for xlσ1R-C179, n = 3 for xlσ1R-C203. “After modification”: n = 6 for xlσ1R WT, n = 4 for xlσ1R-C179, n = 4 for xlσ1R-C203. Two-tailed Student’s t-test was performed between the groups of “Before modification” and “After modification”. The p values are provided in a Source Data file. **p < 0.01. For panels b, c, e, f, data are presented as mean + SD in the bar graphs, which are overlaid with corresponding data points shown as white dots. Source data are provided as a Source Data file.
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Fig. 5. The crystal structure of xlσ1RC179/C203-S1RA.a Superposition of one protomer of xlσ1RC179/C203-S1RA (in green), xlσ1Ropen-endo (in blue), and xlσ1Rclosed-S1RA (in orange), viewed perpendicular to the membrane from the membrane side. b The close-up view of the superposition of one protomer of xlσ1RC179/C203-S1RA (in green) and xlσ1Rclosed-S1RA (in orange), viewed parallel to the membrane. Cys179, Cys203, and the ligand, S1RA, are displayed in sticks.
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