Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual

Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments. These protocols are particularly tailored for cases where the size and molecular weight of the proteins align closely with those of the lipid islands (nanodiscs). While designed for single-span bitopic membrane proteins, these protocols can be easily extended to proteins with multiple transmembrane domains. The insights presented have broad implications across diverse fields, including biophysics, structural biology, and cryogenic electron microscopy (cryo-EM) studies. Key features • Overview of the sample preparation steps from protein expression and purification and reconstitution of membrane proteins in nanodiscs, as well as biobeads and lipids preparation. • Focus on single-span bitopic transmembrane proteins. • Includes protocols for validation procedures via characterization using biochemical, biophysical, and computational techniques. • Guide for cryogenic electron microscopy data acquisition from vitrification to image processing.