A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes

Abstract

The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipidanchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained using in meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that the in meso method also works with soluble proteins should not be overlooked. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.

Crystal structure of the adenosine A 2A receptor bound to an antagonist reveals a potential allosteric pocket

“…To understand the structureactivity relationship around the chemical series for the A 2A R and facilitate further optimization, we sought to obtain the crystal structure of the A 2A R in complex with Cmpd-1. Although a number of crystal structures of A 2A R bound to various antagonists and agonists have been reported (7)(8)(9)(10)(11)(12)(13)(14) using lipidic cubic phase (LCP) methods (15,16), we were unable to obtain high-quality crystals of A 2A R bound to Cmpd-1. As an alternative approach we obtained a 3.5-Å resolution crystal structure of A 2A R in complex with Cmpd-1 by using a BRIL fusion construct and performing the crystallization in vapor-phase diffusion in the detergent lauryl maltose neopentyl glycol (LMNG).…”

“…The LCP method for membrane protein crystallogenesis (15,16) has been used successfully to crystallize a number of GPCRs over the past several years and has become the method of choice; this is exemplified by the fact that 38 of the 39 GPCRs with structures reported have been successfully crystallized in LCP. Therefore, we initially attempted to crystallize A 2A R in complex with Cmpd-1 in LCP.…”

An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

“…Currently, amphipols gained significant popularity in the Cryo-EM field [37][38][39] but also in solution NMR studies [40,41]. For the X-ray crystallography field, it has been shown that amphipols are compatible with lipidic cubic phases [42], thus currently amphipols can only be used for in meso crystallization [43], similarly to SMALPs. There are several other approaches developed (and being actively developed) for the stabilization of membrane proteins, including nanodiscs [44], calixarens [45], fluorinated surfactants [46] and others, but they are beyond the scope of this mini-review.…”

A Molecular Pharmacologist’s Guide to G Protein–Coupled Receptor Crystallography

“…Nevertheless, the majority of GPCR structures have been solved using LCP crystallization (for a comprehensive review, see Caffrey, 2015). In this method, the receptor is crystallized in a lipidic environment instead of from a detergent solution.…”

“…Furthermore, the discovery of new detergents for protein solubilization (Chae et al, 2010;Cho et al, 2015), the development of novel crystallization techniques such as lipidic cubic phase (LCP) crystallization (Caffrey, 2015), and key advances in several aspects of crystallography, such as microcrystallography , X-ray free electron lasers (XFELs), and serial femtosecond crystallography (SFX) have also contributed decisively to the field of GPCR structural biology.…”