Analysis of the movements in the R402C mutant after the MD simulation. (A) Structural deviations observed in the MD simulations. Plots of the RMSD of the Cα atoms from the initial structures of NTD RYR1s (WT, C36R; AB domain mutation, G249R; buried and R402C/H; AC domain mutation) as a function of time are shown. Calculations of each RMSD were performed using MD trajectories from the representative result. The trajectory during 20–50 ns (gray horizontal bars) was analyzed for hydrogen bonds/salt bridges (see Fig. 4, B–F). (B) Superimposition of the monomer of the WT (yellow) and R402C mutant (pink) after 50 ns of the MD simulation. The result shown here is a representative of 20 calculations. The BC domains rotated 14.3 degrees with respect to the A domain in the R402C mutant. The average rotation angle is shown in Fig. S3 E. The rotation axis is indicated by a black line. (C) Superimposition of the BC domains of the WT and R402 mutants after the simulation are shown with either structure almost overlapped. For simplicity, the upper part of the C domain is deleted. (D–F) Closeup view around residue 402. Colors of domains are the same as shown in Fig. 1. Dashed lines represent hydrogen bonds/salt bridges. (D) Crystal structure of the WT NTD (PDB accession no. 2XOA). R402 in the C domain forms hydrogen bond(s) with D61 in the A domain. Thus, through R402, the C domain has a connection with the A and B domains. (E) The same view after 50 ns of MD simulation of the WT NTD. The hydrogen bonds/salt bridges via R402 are tight and are maintained even after 50 ns of the simulation. D61 forms a hydrogen bond with R283 in the B domain (blue dotted line) after 50-ns simulation. (F) The same view after 50 ns of the MD simulation of the R402C mutant NTD. C402 cannot form a hydrogen bond with D61.