Cryo-EM analysis of a membrane protein embedded in the liposome.
Proc Natl Acad Sci U S A
; 117(31): 18497-18503, 2020 08 04.
Article
em En
| MEDLINE
| ID: mdl-32680969
ABSTRACT
Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Escherichia coli
/
Proteínas Associadas à Resistência a Múltiplos Medicamentos
/
Escherichia coli
/
Lipossomos
/
Proteínas de Membrana
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article