Comparison of the initial and follow-up design strategies. The difference in free energy of NKG2D binding relative to wild-type MICA is shown for each MICA mutant tested by SPR, from affinities determined at equilibrium. The RosettaDesign score relative to wild-type is plotted on the x-axis. MICA mutants in the initial design set (internal stabilization or destabilization; Table 1) are shown as open diamonds. MICA mutants from the follow-up design set (significant internal destabilization; Table 2) are shown as filled diamonds.