Volume 112, Issue 8, Pages 1645-1653 (April 2017) Mapping Ryanodine Binding Sites in the Pore Cavity of Ryanodine Receptors  Van A. Ngo, Laura L. Perissinotti, Williams Miranda, S. R. Wayne Chen, Sergei Y. Noskov  Biophysical Journal  Volume 112, Issue 8, Pages 1645-1653 (April 2017) DOI: 10.1016/j.bpj.2017.03.014 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 A snapshot of Ryd in the cavity of RyR during equilibration. (A) Trans-membrane domain of RyR1 (PDB:3J8H) is imbedded in a DMPC lipid bilayer (magenta sticks); the water medium is also shown (colored in pink). PDB and structure files of Ryd are provided in the Supporting Material. (B) Shown here is the water-pore cavity with S6 bundles that flank above the ryanodine. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Normalized distribution of interaction energies between Ryd and the rest of the system from REST2 simulations. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Angular distributions for Ryd binding. The value n→ is the unit vector between the geometric center of the pyrrolic ring and the geometric center (R0) of the heavy atoms without the ring from REST2 simulations. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Unnormalized distributions of angle φ of a vector projected on xy-plane with respect to the x axis at different temperatures. Such a vector can be n→ (see definition in Fig. 3). The other four projected vectors are drawn between R0 and the centers of four aromatic rings of residues F4921 from REST2 simulations. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 PMF for the movement of Ryd from the lumen through the SF and pore helices (PH) to the cavity obtained from bidirectional pulling simulations (see Materials and Methods for definition of Δz). The convergence analysis is provided in Fig. S5. The uncertainty (shaded areas) of the PMF is, at most, 0.5 kcal/mol. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 A snapshot showing Ryd interacting with the SF and lumen. F4921 residues are colored in magenta. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 (a) Normalized distribution of angle θ during the entering of Ryd into the cavity from the lumen (Δz from ∼−22 to 3 Å). (b) Shown here are the unnormalized distributions of φ after Ryd passes the free-energy barrier (Δz from ∼−5 to 3 Å). To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 Relative distance between Ryd and the center of mass of all α-carbon atoms of RyR at different temperatures. (a) Wild-type. (b) Mutant. Negative values indicate that Ryd is further away from the constriction point, where the glutamine residues are located. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 9 (a) Interaction energy between Ryd and four amino acids Q4933 and A4933 in the WT and mutated structures. (b) Number of water molecules in the cavity counted during the entering of Ryd between residues G4894 and Q4933, which determine the two end points of the cavity along the z axis. To see this figure in color, go online. Biophysical Journal 2017 112, 1645-1653DOI: (10.1016/j.bpj.2017.03.014) Copyright © 2017 Biophysical Society Terms and Conditions