Cryo-EM structures of prothrombinase free and bound to prothrombin. (A) The structure was solved at 5.3-Å resolution and shows the 2 proteins of the prothrombinase complex, fVa and fXa, in surface representation. Images including the nanodiscs are given in supplemental Figures 1 and 2. The architecture of the A1 (smudge), A2 (pale green), A3 (lime green), C1 (lime), and C2 (lemon) domains is solved in its entirety, except for the N-terminal ¹⁵⁴⁶SNNGNRRNYY¹⁵⁵⁵ sequence of the A3 domain immediately downstream of the site of thrombin activation at R1545.⁵ The overall arrangement of fVa is very similar to that of the free form solved recently.³⁵ The architecture of fXa is fully resolved and provides a picture of the complete arrangement of the constitutive Gla (raspberry), EGF1 (dark salmon), EGF2 (salmon), and protease (deep salmon). The bound fXa features an overall conformation with the domains not vertically aligned but bent over the EGF1-EGF2 junction, as documented in several X-ray structures,^28,29 and additionally 90° at the EGF1-Gla domain junction (supplemental Figure 4C). Also shown are the sites of fVa inactivation by activated protein C at R306 and R506 (red) and the site of thrombin activation at R709 (magenta). The enzyme is positioned in the complex along the A2, A3, and C1 domains of fVa, with the Gla domain aligned with C1 domain on the plane of the nanodisc (see supplemental Figure 1A). A segment of the A2 domain lowers on the fXa to fix the protease domain for optimal interaction with an incoming substrate (see Figure 2 for details). (B) The structure was solved at 4.1-Å resolution and shows fVa and fXa in the same arrangement found on nanodiscs (see panel A), which supports the ternary complex as a genuine representation of the prothrombin-prothrombinase complex. The constitutive domains of prothrombin are colored in sand (Gla domain), pale yellow (EGF1), yellow (EGF2), and orange yellow (protease domain, PD), with the Gla domain aligned with the homologous domain of fXa and the C1 and C2 domains of fVa for interaction with membranes. Prothrombin faces fXa in the complex and is positioned along the A2, A1, and C2 domains of fVa. Visible in this orientation is the site of activation at R271 (blue), located above the protease domain of fXa, but not R320 that inserts into the active site of fXa (see Figure 2 for details). The overall architecture of prothrombin is similar to that of the closed form^31,33 (supplemental Figure 4A).