Fig. P1.

(A) A ribbon representation of the nonactivated, resting-state Notch NRR multidomain structure is shown on the right (LNR modules are represented by shades of purple, and the HD domain is in blue). The NRR is attached to AFM components via specific chemistry at its termini. The diagram shows how we used AFM to apply force and stretch the molecule. At an early stage, the first two LNR modules peel away from the HD domain to produce the conformation indicated on the left, in which the S2 site (indicated by a yellow star) becomes accessible to cutting by TACE. (B) The percentage frequency of cleavage events that we observed during AFM experiments. Essentially, significant cleavage rates in the presence of functional TACE are observed when force is applied to Notch NRR, demonstrating S2 site accessibility.