Issue link: https://resources.nanotempertech.com/i/1526380
2 A P P L I C A T I O N N O T E Cryo-EM structural determination has vastly expanded the number and complexity of atomic- resolution structures available, facilitating a better understanding of mechanisms including cellular regulation that guide therapeutic development. Previously difficult-to-crystallize targets such as membrane proteins have become more feasible for structural analysis, despite their amphipathic nature, large size, and conformational flexibility. However, high-resolution cryo-EM studies depend on preparing grids with pure, monodisperse, well-folded proteins, which is still a significant hurdle to overcome. This challenge is especially pronounced for membrane proteins, as classical methods for solubilization and purification are laborious, and contain many potentially destabilizing steps (1) . Cube Biotech capitalizes on their expertise as a protein expression and purification service provider to optimize a wide variety of membrane protein classes. The company has found that their 100 per- cent detergent free NativeMP TM Platform, featuring a variety of synthetic copolymers derived from 5 different chemical backbones (SMA, DIBMA, AASTY, Ultrasolute TM Amphipol and Cubipol), are ex- cellent reagents for solubilizing and stabilizing membrane pro teins of all classes for cryo-EM based structure determination. The purified copolymer nanodiscs are composed of membrane proteins embedded within native cell membrane phospholipids. These complexes are held together by a synthetic copolymer ring, which offers improved stability when solubilizing membrane proteins for cryo-EM investigation. Since Cube Biotech receives a large number and variety of membrane proteins for cryo-EM optimization, it is important to have high-throughput methods for optimizing which synthetic copolymers are best suited to the target proteins. They have found that sizing data provided by dynamic light scattering (DLS) measurements, along with conformational stability data from nano differential scanning fluorimetry (nanoDSF) obtained on the Prometheus Panta are incisive quality- control parameters for the copolymer screen. High-throughput optimization of this screening process also benefits academic researchers, by significantly reducing the amount of time and required material to identify optimal copolymers. Introduction