BIVV001, a stabilized FVIII/VWF-DʹD3 complex, was an ideal candidate for single particle, cryo-electron microscopy. (A) N to C terminal linear domain layout comparing WT FVIII (top) to BIVV001 (bottom). Large structured domains of FVIII are in blue, with intervening acidic peptides in yellow, and B domain in white. In BIVV001, XTEN insertions are in gray, Fc in green, and Dʹ and D3 in red and orange, respectively. Positions of C1099A and C1142A mutants to prevent VWF dimerization are shown. Thrombin cleavage sites are depicted by scissors, with posttranslational modification sites indicated by spheres: S, tyrosine sulfation; N, N-linked glycosylation. (B) Illustration of FVIII/VWF-D′D3 complex for WT FVIII and endogenous VWF. (C) Illustration of VWF-D′D3 complex within BIVV001. Arrows depict likely interaction sites based on previously reported biochemical data.