Negative-stain EM characterization of the αIIbβ3 ectodomain in complex with PT25-2 Fab. (A) Raw image of negatively stained αIIbβ3 ectodomain. Some integrins in the fully bent-closed conformation are indicated by dashed circles, whereas some integrins in more extended conformations are indicated by ovals. (B) Selected 2D class averages of negatively stained αIIbβ3 ectodomain by itself. Average 1 represents the fully bent-closed conformation. The diagram in panel E illustrates the ∼45° angle between the head and leg domains. Averages 2 to 5 show increasingly extended closed conformations. Average 6 shows a side view, and averages 7 and 8 show face-on views of the fully extended-closed conformation. (C) Raw image of negatively stained αIIbβ3 ectodomain in complex with PT25-2 Fab. Some integrins with a bound Fab are indicated by ovals. (D) Selected 2D class averages of negatively stained αIIbβ3 ectodomain in complex with PT25-2 Fab. Average 1 shows the complex in the most compact conformation. The diagram in panel F illustrates the ∼80° angle between the head and leg domains, and the arrowhead points to the PT25-2 Fab that prevents a closer association of the leg domains with the headpiece. Averages 2 to 7 show increasingly extended closed conformations of the PT25-2 Fab-bound αIIbβ3 ectodomain. Average 8 shows a side view of the fully extended-closed conformation. The scale bars in panels A and C are 50 nm, and the side length of the individual class averages in panels B and D is 43.2 nm.