Figure 1. Sites, agonists, and cycle. (a) Ligand binding sites. Side view of an acetylcholine binding protein, homologous to the AChR extracellular domain (PDB ID: 3WIP ; Olsen et al., 2014 ). Sites are at subunit interfaces. The agonist (ACh, More about this image found in Sites, agonists, and cycle. (a) Ligand binding sites. Side view of an acet...

Figure 2. Binding site structures (x-ray) . (a) AChBP with ACh (top; PDB ID: 3WIP ; Olsen et al., 2014 ) or nicotine (bottom; PDB ID: 1UW6 ; Celie et al., 2004 ). Dashed lines are H-bonds; red dot is a structural water. (b) α4β2 AChR with More about this image found in Binding site structures (x-ray) . (a) AChBP with ACh (top; PDB ID: 3WIP ...

Figure 3. Binding site structural parameters. Pocket volume was calculated as that of the pyramid formed by joining the centers of the five aromatic rings (front face, white). Distances are between the agonist’s principal nitrogen ( Fig. 1 b ) More about this image found in Binding site structural parameters. Pocket volume was calculated as that o...

Figure 4. RMSD of the protein backbone. The backbone equilibrates within ∼10 ns. Most of the residual fluctuations are from loop F. Figure 4. RMSD of the protein backbone. The backbone equilibrates within ∼10 ns. Most of the residual More about this image found in RMSD of the protein backbone. The backbone equilibrates within ∼10 ns. Mos...

Figure 5. Pocket volume and d_x . Resting, brown and active, green. (a) Top: Pocket volume. With all agonists, α−γ is the smallest (^AR and ^AR*). At all sites, ^AR* is smaller than ^AR (all agonists) and pocket volume is smallest with TMA. Bottom: More about this image found in Pocket volume and d_x . Resting, brown and active, green. (a) T...

Figure 6. Water density. The number of water molecules was counted in spheres of radii 5, 10, and 20 Å, with the origin at the pocket center (α−γ). There is no significant difference between ^AR and ^AR* conformations or between agonists. The 20 Å More about this image found in Water density. The number of water molecules was counted in spheres of rad...

Figure 7. Resting versus active α−γ pocket metrics . Distances are ^AR*/^AR ratios and angles are ^AR* – ^AR differences. Left and center columns: Metrics that differ significantly between ^AR* and ^AR. For ACh-class agonists (colored bars), in R* More about this image found in Resting versus active α−γ pocket metrics . Distances are ^AR*/...

Figure 8. Energy–structure correlations. (a) Linear correlations between experimental binding energy and binding site metrics (ACh, CCh, TMA, and Cho; all sites; ^AR and ^AR*). The highest correlation is with d_x. (b) Pearson’s correlation More about this image found in Energy–structure correlations. (a) Linear correlations between experimenta...

Figure 9. Resting versus active α−γ neurotransmitter binding site. (a) For all agonists, the active pocket (^AR*, green) is smaller than the resting pocket (^AR, brown; Fig. 3 ). (b) ^AR* versus ^AR with ACh. In the active state, the agonist’s More about this image found in Resting versus active α−γ neurotransmitter binding site. (a) For all agoni...

Figure 10. Affinity, efficacy, and efficiency . In each plot, y-axis values are free energies from electrophysiology experiments and x-axis values are distances from equilibrated structures. Open symbols, ^AR; closed symbols, ^AR*. (a) Distance More about this image found in Affinity, efficacy, and efficiency . In each plot, y-axis values are free e...

Figure 11. CRCs. Symbols are from electrophysiology experiments, and solid lines are calculated from d_x values. Inset: CRCs for two agonists that were not used in the energy–d_x correlation ( Fig. 10 a ). See Fig. 1 b for agonist structures. More about this image found in CRCs. Symbols are from electrophysiology experiments, and solid lines are ...

Figure 1. Cysteine reactive cross-linkers dimerize subunits. (A) Model of the hP2X1R showing the location of the G30C mutation (black spheres). The three subunits are shown in gray, light blue, and light pink. (B) Chemical structures of BMB More about this image found in Cysteine reactive cross-linkers dimerize subunits. (A) Model of the hP2X1R...

Figure 2. Cross-linking can be prevented by cysteine residue blockade. (A) Representative blots from HEK293F cells transfected with hP2X1 C349A and G30C C349A, treated with 3.2 U/ml apyrase only, apyrase then BMB, or apyrase then NEM followed by More about this image found in Cross-linking can be prevented by cysteine residue blockade. (A) Represent...

Figure 3. Specific amino-terminal cysteine residues form cross-links. (A) Representative blots from HEK293F cells transfected with hP2X1 C349A, R25C, N26C, K27C, K28C, V29C, or G30C treated with 3.2 U/ml apyrase, then BMB or DMSO (apyrase only). More about this image found in Specific amino-terminal cysteine residues form cross-links. (A) Representa...

Figure 4. Specific carboxyl-terminal cysteine residues form cross-links. (A) Representative blots from HEK293F cells transfected with hP2X1 C349A H355C, I356C, L357C, P358C, K359C, or R360C treated with 3.2 U/ml apyrase, then BMB or DMSO More about this image found in Specific carboxyl-terminal cysteine residues form cross-links. (A) Represe...

Figure 5. Specific amino and carboxyl-terminal cysteine residues form cross-links with ATP pretreatment. (A) Histogram showing the average percentage dimer present for R25C to G30C tested with all three cross-linkers in the presence of 300 µM More about this image found in Specific amino and carboxyl-terminal cysteine residues form cross-links wit...

Figure 6. Visualization of cross-links on structural models. (A) TM1 of the hP2X1R in the closed state and residues 25–29 mutated to cysteines with BMB (10.9 Å) as cross-linker. (B) Cartoon representation of TM1 and N-terminal region (one More about this image found in Visualization of cross-links on structural models. (A) TM1 of the hP2X1R i...