Using Hybrid Methods To Refine A Complete Atomic Structure Of The Human Integrin Alpha Iib Beta 3 Ectodomain Into A Low-Resolution Electron Microscopy Map Of The Intact Receptor In Nanodiscs

The platelet integrin αIIbβ3 receptor plays a crucial role in thrombosis and hemostasis. A full-length atomic structure of the receptor is required to fully understand ligand binding and signaling. Guided by a 20.5 A low-resolution electron microscopy (EM) map of the intact receptor in phospholipid bilayer nanodiscs, we used a combination of molecular dynamics flexible fitting (MDFF) and steered MD methods to refine a complete atomic structure of the human αIIbβ3 ectodomain into the EM maps. The complete ectodomain was built using the closed αIIbβ3 crystal structure and ab-initio methods to reconstruct missing loops. Several landmarks guided the MDFF, including binding of mAbs to specific domains as revealed by negative stain images. The characteristic shape of the αIIb β-propeller and the mAb binding sites furnished the basis for fitting the αIIb β-propeller and the β3 βI and PSI domains into the map. Reasoning that the electron density adjacent to the bilayer must fit distal segments of the integrin legs, we steered the corresponding crystallographic regions towards the section of the map immediately above the nanodisc. The adjacent β3 EGF-4 and EGF-3 domains were steered towards the higher density regions in the lower part of the map, whereas a final MDFF run allowed optimal fitting of the β3 EGF-1 and EGF-2, and the αIIb thigh domains into the remaining electron density. Our predicted model structure of the αIIbβ3 ectodomain shows that the headpiece is oriented away from the lipid bilayer and, in sharp contrast to the crystal structure, that the lower legs are not straight, parallel, and adjacent; rather they show definite bends and join only when near the bilayer.