Project Summary for Parent Grant R01GM132329 Membrane proteins are essential in living organisms. Protein-lipid interactions are critical for membrane protein structural and functional integrity. High-resolution membrane protein structures are in high demand for understanding their biology and drug development based on structure. Extraction of membrane proteins from cell membranes is frequently required for biochemical and biophysical research. Currently, detergents are the most often used method for extracting membrane proteins. Detergents have several downsides, including the destruction of lipid bilayers and the removal of essential lipid molecules from membrane proteins. We are creating a detergent-free native cell membrane nanoparticles technology for membrane protein research. The native cell membrane nanoparticles system is made up of three parts: 1) Polymers that are membrane-active. 2) Methods for creating native cell membrane nanoparticles. 3) Cryo-EM single particle investigation of native cell membrane nanoparticles. We suggest creating a system that is applicable to both bacterial and eukaryotic membrane proteins. Specific Aim 1 focuses on building techniques to create native cell membrane nanoparticles utilizing our chosen model membrane proteins and constructing a membrane-active polymer library. We have created four prototype membrane active polymers; we intend to expand the active membrane polymers utilizing both commercially available styrene-maleic anhydride copolymers and polymers made in laboratories using similar procedures. Specific Ami 2 focuses on creating procedures for determining the high-resolution structure of selected bacterial model membrane proteins. The third specific aim is to create procedures for high-resolution structures of selected eukaryotic model membrane proteins. The proposed study is interesting because it offers a novel detergent-free method for investigating membrane proteins in their native lipid context. As our first data show, structural knowledge of protein-lipid interactions for membrane proteins is critical for understanding the structure and function of membrane proteins. This approach has the potential to revolutionize the area of membrane protein research and have a significant impact on membrane protein structure-based medication discovery and development.