Analysis of molecular stiffness. (A and B) Histograms (gray bars) and their dual Gaussian fits (green and pink solid curves) of the indicated numbers of stiffness of α_Lβ₂–ICAM-1 molecular complexes measured by the stretch method in Ca²⁺Mg²⁺ (A) and Mn²⁺ (B). The x-axis value with minimum overlap between the two Gaussian distributions (0.5 pN/nm) was used to set a threshold (blue dash line) to differentiate soft (k_mol < 0.5 pN/nm) and stiff (k_mol > 0.5 pN/nm) α_Lβ₂–ICAM-1 molecular complexes. (C) Relative stiffness (to the value in Mn²⁺) of the α_Lβ₂–ICAM-1 complex measured using the thermal fluctuation method at zero force in Ca²⁺Mg²⁺ or in Mn²⁺ with or without 1 µM XVA143 (means ± SEM of indicated numbers of measurements). (D) Stiffness (means ± SEM of >50 measurements) of α_Lβ₂–ICAM-1 molecular complexes measured by the stretch method in Mn²⁺ plus increasing concentration of XVA143 in solution (points directly connected by solid lines). Stiffness of α_Lβ₂–ICAM-1 complexes in Ca²⁺Mg²⁺ and of α_Lβ₂–KIM127 complexes in Mn²⁺, both in the absence of XVA143, are shown as dashed lines (indicated). (E) Histograms of the indicated numbers of α_Lβ₂–ICAM-1 complex stiffness measured before up or down events from the ramp phase using the stretch method. (F) Relative (to the respective values of the putative extended α_Lβ₂–ICAM-1 complex) molecular stiffness before up and down events measured by the stretched method (left), and those before and after up and down events measured by the thermal method (right). Error bars represent SEM of >50 measurements.