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Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a- x . The results demonstrated that NCMNP2a- x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize Bc TSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a- x might have broad applications in membrane protein research., Competing Interests: Y. G. and T. K. H. T. are inventors of NCMNP2a-x (patent pending)., (This journal is © The Royal Society of Chemistry.)