Figure 5.

Ensemble refinement of H212R apo-PhuS by ST, HDXer, and CD. (A) Plot of the average RMSD (Å) of the C_α at each residue for a 5-μs-long conventional MD simulation of H212R apo-PhuS simulation (black) and the six combined ST simulations of H212R apo-PhuS (red). (B) Decision plot resulting from the reweighting of the combined H212R apo-PhuS ST runs to the target H212R apo-PhuS HDX-MS data. The apparent work obtained using selected γ of 0.9 is indicated with a red dashed line. (C) Violin plots of the seven significantly upweighted clusters ranked in order of average change in weight (log(Ω_f/Ω_i)). The tails represent the minimal and maximal weight in the cluster, the thick dashed lines indicate the mean change in weight, and the thin dashed lines bracket 50% of the population. The change in percent α-helicity (Δα_h) observed between each cluster and the holo-PhuS computational simulations (blue dots) is shown alongside a weighted average for all clusters (blue dot-dashed line). The red dotted line indicates the experimental Δα_h. (D) The KDE plot generated from the seven significantly upweighted clusters using the distance between the C_α of Arg212 and Phe114 (Å) and the percent α-helicity in the α6,7,8 region. Each number represents the average coordinate of one of the seven significantly upweighted clusters, numbered according to its corresponding average upweighting rank (most upweighted to least upweighted). The scale bar represents the probability desnsity at each point. The inset shows the Arg212 residue is within 2.2 Å of Glu318 (sticks), an interaction observed in all but clusters 4 and 6.