Subtomogram averaging of protein structures in isolated HEK293 cell plasma membranes

Abstract

The plasma membrane and its associated proteins together are key to the function and disfunction of many vital cell processes such as adhesion, sensing/signaling, endocytosis/exocytosis. To better understand their function and treat their disfunction with precision, we need high-resolution structures of membrane-associated molecular components in their native context. Cryo-electron tomography (cryoET) is a rapidly evolving structural determination technique capable of resolving proteins and protein complexes to ångström-level resolution. However, high-resolution reconstruction of membrane-associated proteins and organelles in situ has been met with obstacles including the necessity for ultrathin (<200 nm thick) sample preparation. Here, we isolate plasma membranes using a technique called “unroofing” immediately prior to sample vitrification. This produces 50-200-nm-thick vitreous samples containing the plasma membrane, the actin cortex, and the myriad of other protein complexes associated with the membrane. To verify this technique as a tool for membrane protein structure determination, we are performing a quantitative assessment of the preserved resolution using subtomogram averaging. We are currently working to utilize and modify existing subtomogram averaging tools to identify, segment, process, and refine objects of interest within reconstructed tomograms. We compare our reconstructions with known protein structures. This ongoing work is part of an effort aimed to develop a correlative pipeline for visualizing the proteome of the eukaryotic plasma membrane with efficiency and precision.