Extended Data Fig. 4: Cross-linking mass spectrometry analysis of P-Rex1.

BS³ cross-linking constraints and Circos plots of cross-linking mass spectrometry are shown for a. the isolated DH-PH domains, b. the DH-PH^T4L domains, c. the DH-PH-DEP1 domains, d. the DH-PH-DEP1^T4L domains, e. full-length wild-type (WT) P-Rex1, and f. the full-length ^ΔN40P-Rex1^{T4L, Δloop} construct utilised for cryo-EM studies. Loop refers to residues 1119-1211. Lysine Cβ atoms are shown as blue spheres with blues lines indicating a compatible constraint (<30 Å between lysine Cβ atoms) and red lines indicating an incompatible constraint (>30 Å between lysine Cβ atoms). Constraints are mapped onto active (left) or autoinhibited (right) models of the DH-PH-DEP1 domains. Cross-links are in excellent agreement with the closed conformation observed in our DH-PH-DEP1 crystal structure. These data indicate that the closed conformation is stably populated in solution in the presence or absence of T4L. Conversely, cross-links frequently exceeded the allowable constraint distance when modelled on the active conformation. Full-length P-Rex1 displays a similar cross-linking pattern in the absence or presence of T4L (e-f). Interestingly, incompatible constraints (red lines) cluster across the DEP1–DEP2 linker region in full-length P-Rex1 indicating potential conformational flexibility across this interface. Dashed line (black) indicates position of an intrinsically disordered loop (IDL) and the FDR is indicated for each dataset.