Cryo-EM structures. fV (A-C) and fVa (D-F) structures are shown. The 2 proteins are 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]. The structure of fV (A-C) was solved at 3.3 Å resolution and features the C domains aligned edge to edge, forming a platform involved in membrane binding and upon which the A domains rest side by side, arranged around a pseudo-3-fold axis. The A1-A2-A3-C1-C2 domain assembly is resolved in its entirety. The sites of thrombin activation at R709 and R1545 (magenta) are clearly visible in the A2 and B domains and are exposed to solvent for proteolytic attack. The sites of APC cleavage at R306 and R506 (red) are 75% buried. The exact orientation of the side chains at the sites of cleavage by thrombin and APC were assigned with sufficient confidence in the context of the resolution obtained (shown also in Figures 3B-C and 4). The structure also reveals the spikes in the C domain responsible for membrane docking (olive) and the epitopes of binding for fXa (orange), APC (blue), and prothrombin (gray), which occupies separate segments in the C-terminal of the A2 domain and the C2 domain (arrows labeled proT). Most of the fXa epitope is buried under the short segment ³⁷³DESF³⁷⁶ (asterisk) of the A2 domain in both fV (C) and fVa (F). A rearrangement of this segment appears necessary to fully expose the epitope upon assembly of prothrombinase. The B domain is very dynamic; only 14 residues are resolved in the connection to the A2 domain (⁷¹⁰SFRN⁷¹³ segment) and the A3 domain (¹⁵³⁶PDNIAAWYLR¹⁵⁴⁵ segment). The structure of fVa (D-F) was solved at 4.4 Å resolution and is more disordered than that of fV, with fewer (1181 of 1360) residues resolved in the A1 (294 of 316), A2 (294 of 393), A3 (295 of 332), and C2 (139 of 160) domains. Overall, the arrangement of the A1-A2-A3-C1-C2 domain assembly is similar to that of fVa (RMSD = 2.26 over 972 Cα atoms). The disorder in fVa affects the spikes in the C2 domain (D) and the entire epitope of prothrombin binding (gray) that are missing in the density map (E-F). Structuring of the prothrombin epitope may be necessary to assemble a functional prothrombinase-prothrombin complex. Importantly, the sites of APC cleavage at R306 and R506 leading to fVa inactivation are 60% exposed to solvent (E-F), unlike what is seen in fV (B-C). The structures also provide context for the functional consequences of posttranslational modifications and mutations in the C1 and C2 domains that reduce binding of fV to phospholipid membranes and perturb its cofactor activity. The site of glycosylation at N2181 and residue A2086 are clearly visible on the surface of the C2 domain, but residue W1920 is buried under Y1903 in the C1 domain (A,D).