Cryo-EM structure of fV short. The cryo-EM structure of fV short was solved at atomic (3.2 Å) resolution and reveals all residues (1493 total), including the short (133 residues total) B domain, of the A1-A2-B-A3-C1-C2 assembly. The protein is rendered in surface representation with the constitutive domains colored in wheat (A1), pale green (A2), light blue (B), pale yellow (A3), light pink (C1), and pale cyan (C2) and features an overall organization similar (rmsd = 2.22 Å over 1134 Cα atoms) to that of fV reported recently.¹² (A) The C domains align edge-to-edge to define a membrane-binding platform that supports the A1 and A3 domains side by side, with the A2 domain on top of them. The A2 domain houses the gate (blue; ⁶⁹⁶YDYQNRL⁷⁰²) and the lid (orange; ⁶⁷²ESTVMATRKMHDRLEPEDEE⁶⁹¹) that play an important role in the prothrombinase complex.¹⁶ The sites of thrombin activation at R709 and R1545 are clearly visible in the A2 and B domains, as are the sites of APC cleavage at R306 and R506. The B domain (133 residues total) is resolved in its entirety and stretches across the entire width of the protein, from R709 to R1545, making contacts with the A1, A2, and A3 domains (Table 2) and being suspended over the C1 and C2 domains. (B and C) After reaching the opposite side of fV, near the site of thrombin cleavage at R1545, the B domain loops over the A3 domain in the back of the protein, pointing upward toward the lid in the A2 domain. Residues of the HPC4 tag (supplemental Figures 1C and 3) were removed from this rendering for clarity.