Cryo-electron tomography pipeline for plasma membranes
Willy W. Sun, Dennis J. Michalak, Kem A. Sochacki, Prasanthi Kunamaneni, Marco A. Alfonzo‐Méndez, Andreas M. Arnold, Marie‐Paule Strub, Jenny E. Hinshaw, Justin W. Taraska
Abstract
Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells.