Atomic-level Structural-omics of Platelet Membrane Proteins via Multi-scale Structural Biology Approaches - PROJECT SUMMARY Membrane proteins play a critical role in platelet function by relaying extracellular signals outside in. Changing the quantity or quality of these proteins can alter platelet physiology and impact health. The native membrane environment provides additional cues that regulate the protein structure and mechanism of action. Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by solving high-resolution structures of membrane proteins and large macromolecules from homogeneous samples. A recent breakthrough in data processing, the Build and Retrieve (BaR) methodology, performs in silico purification from large heterogenous cryo-EM datasets, making it feasible to obtain protein structures from crude preparations in their native environments. Using Cryo-Focused Ion Beam (cryo-FIB) on whole cell sample preparation provides opportunities to directly visualize macro-complex in situ. How the native environment regulates platelet function at the near-atomic level will be uncovered by combining these technical advances. The preliminary studies using cryo-EM coupled with BaR focused on proteins that are ~200kDa. It solved the first unmodified integrin αIIbβ3 structure in inactivated and intermediate states at 2.76Å and 2.49Å with metal ions, N-linked glycans, and a ligand preserved. A third structure identified a novel dimer conformation of αIIbβ3 at 2.61Å, which potentially indicates an unknown αIIbβ3 self-regulatory mechanism in the native environment. The scientific goal of this application is to solve membrane protein structures from resting and activated platelets from healthy donors without artifacts due to purification processes. Aim 1 (K99 phase) will focus on extending the preliminary αIIbβ3 data to determine the in-situ dynamics of integrin activation. Aim 2 (R00 phase) will focus on building the structural atlas of platelet membrane proteins using the combination of single-particle cryo-EM, in-situ cryo-EM, and cryo-ET with the BaR method. This aim will start with a specific focus on mitochondrial supercomplex and ATP synthase. Ultimately, solving the structures of membrane proteins in their native environment will reveal conformations and interactions that govern protein function. Obtaining these insights will pave the way for significant advancements in disease orientation, disease diagnostics, and therapeutic interventions in patients with platelet disorders. The career goal in this application is to set the trajectory for the candidate’s independent research program to conduct highly innovative research to bridge the hematology and structural biology fields. The candidate will acquire experimental training for in-situ cryo-EM studies and cryo-ET during the K99 phase. Working with the mentoring team, this application outlines an intensive training plan to foster the further growth of the core competencies (communication, critical thinking, professionalism, creativity, teamwork) for transitioning the candidate into a well-equipped independent investigator in hemostasis and thrombosis field with a unique combination of research skillsets and a highly innovative and scientifically promising research platform.
