Crystallizing Membrane Proteins Using Lipidic Mesophases

By M. Caffrey, Vadim Cherezov. Nature Protocols, 2009

655 citations recorded by the source corpus.

A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human-engineered β2-adrenergic and adenosine A2A G protein–coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase and for setting up crystallizations in manual mode. Methods for harvesting microcrystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about 1 h.

Subfields: Molecular Biology

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Citations in the corpus

Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor (2012, methodology): “5 parts by weight of a 10:1 mix of monoolein with cholesterol (Sigma) using the two syringe reconstitution metho”
5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology (2018, methodology): “Lipidic Cubic Phase Crystallization The purified 5-HT2C protein in complex with ERG or RIT was screened for crystallization in lipidic cubic phase (LCP) with mixed molten lipid (90% (w/v) monoolein and 10% (w/v) cholesterol) at a protein/lipid ratio of 2:3 (v/v) using amechanical syringemixer (Caffr”
GPCR Network: a large-scale collaboration on GPCR structure and function (2012, methodology): “Crystallization trials are performed using the in meso (LCP) 57 approaches.”
Inward-facing conformation of a multidrug resistance MATE family transporter (2019, methodology): “The crystals of PfMATE in the inward- and outward-facing conformations (6FHZ and 6GWH, respectively) were obtained by the LCP technique (30, 31).”
Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase (2020, methodology): “The receptor was reconstituted in LCP by mixing with a lipid mixture consisting of 90%(w/w) monoolein and 10%(w/w) cholesterol at a ratio of 2 parts protein to 3 parts lipid by volume using a lipid syringe mixer (Caffrey & Cherezov, 2009).”
Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist (2012, methodology): “5-fold weight excess of a 10:1 monoolein:cholesterol mix by the twin-syringe metho”