Lipid Nanodiscs as a Tool for High-Resolution Structure Determination of Membrane Proteins by Single-Particle Cryo-EM.

TitleLipid Nanodiscs as a Tool for High-Resolution Structure Determination of Membrane Proteins by Single-Particle Cryo-EM.
Publication TypeJournal Article
Year of Publication2017
AuthorsEfremov, R. G., C. Gatsogiannis, and S. Raunser
JournalMethods Enzymol
Volume594
Pagination1-30
Date Published2017
ISSN1557-7988
KeywordsAnimals, Bacterial Toxins, Cryoelectron Microscopy, Lipid Bilayers, Lipids, Membrane Proteins, Models, Molecular, Molecular Structure, Nanostructures, Rabbits, Ryanodine Receptor Calcium Release Channel
Abstract

The "resolution revolution" in electron cryomicroscopy (cryo-EM) profoundly changed structural biology of membrane proteins. Near-atomic structures of medium size to large membrane protein complexes can now be determined without crystallization. This significantly accelerates structure determination and also the visualization of small bound ligands. There is an additional advantage: the structure of membrane proteins can now be studied in their native or nearly native lipid bilayer environment. A popular lipid bilayer mimetic are lipid nanodiscs, which have been thoroughly characterized and successfully utilized in multiple applications. Here, we provide a guide for using lipid nanodiscs as a tool for single-particle cryo-EM of membrane proteins. We discuss general methodological aspects and specific challenges of protein reconstitution into lipid nanodiscs and high-resolution structure determination of the nanodisc-embedded complexes. Furthermore, we describe in detail case studies of two successful applications of nanodiscs in cryo-EM, namely, the structure determination of the rabbit ryanodine receptor, RyR1, and the pore-forming TcdA1 toxin subunit from Photorhabdus luminescens. We discuss cryo-EM-specific hurdles concerning sample homogeneity, distribution of reconstituted particles in vitreous ice, and solutions to overcome them.

DOI10.1016/bs.mie.2017.05.007
Alternate JournalMeth. Enzymol.
PubMed ID28779836
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