Elucidating the dynamics of Integrin αIIbβ3 from native platelet membranes by cryo-EM with build and retrieve method.

Platelets fulfill their essential physiological roles sensing the extracellular environment through their membrane proteins. The native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins to be solved from homogeneous samples. Our recent breakthroughs in data processing now make it feasible to obtain atomic-level-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the "Build-and-Retrieve" (BaR) data processing methodology. We applied this iterative bottom-up methodology on resting human platelet membranes for an in-depth systems biology approach to uncover how lipids, metal binding, post-translational modifications, and co-factor associations in the native environment regulate platelet function at the molecular level. Here, we report using cryo-EM followed by the BaR method to solve the unmodified integrin αIIbβ3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.75Å and 2.67Å, respectively. Further, we also solved a novel dimer conformation of αIIbβ3 at 2.85Å formed by two intermediate-states of αIIbβ3. This may indicate a previously unknown self-regulatory mechanism of αIIbβ3 in its native environment. In conclusion, our data show the power of using cryo-EM with the BaR method to determine three distinct structures including a novel dimer directly from natural sources. This approach allows us to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. These data have the potential to enrich our understanding of platelet signaling circuitry.