eIF2B structure predicts activity of ISRIB analogs. (A) GEF activity of assembled eIF2B(βγδε) and eIF2B(α2) in the presence and absence of ISRIB-A19(R,R) and ISRIB-A19(S,S). (B) Stability of decameric eIF2B(δL179A) in the absence of ISRIB (top), presence of ISRIB-A19(S,S) (middle), or presence of ISRIB-A19(R,R) (bottom) as assessed by velocity sedimentation on sucrose gradients. (C) eIF2B GEF activity of assembled eIF2B(βγδε) and eIF2B(α2) containing a δL179A mutation in the presence and absence of ISRIB-A19(R,R) and ISRIB-A19(S,S). (D) Quantification of eIF2B decamer stability gradients plotted as fraction of eIF2B(βγδε) present in each of lanes 1-13. eIF2B (for comparison from data shown in ), eIF2B(βH188A), eIF2B(βH188Y), eIF2B(βH188F) gradients are plotted in the presence (bottom panel) and absence (top panel) of 500 nM ISRIB. (E, F, G) Stability of decameric eIF2B(βH188A), eIF2B(βH188Y), and eIF2B(βH188F) in the presence of ISRIB as assessed by velocity sedimentation on sucrose gradients.