Abstract
The lipid-based bicontinuous cubic mesophase is a nanoporous membrane mimetic with applications in areas that include medicine, personal care products, foods and the basic sciences. An application of particular note concerns it use as a medium in which to grow crystals of membrane proteins for structure determination by X-ray crystallography. At least two variations of the mesophase exist. One is the highly viscous cubic phase, which has well developed long-range order. The other so-called sponge phase is considerably more fluid and lacks long-range order. The sponge phase has recently been shown to be a convenient vehicle for delivering microcrystals of membrane proteins to an X-ray free-electron laser beam for serial femtosecond crystallography (SFX). Unfortunately, the sponge phase approach calls for large amounts of protein that are not always available in the case of membrane proteins. The cubic phase offers the advantage of requiring significantly less protein for SFX but comes with its own challenges. Here, we describe the physico-chemical bases for these challenges, solutions to them and prospects for future uses of lipidic mesophases in the SFX arena.
Highlights
Serial femtosecond X-ray crystallography (SFX) is a relatively new method for collecting crystallographic information on small crystals fed continuously into a free-electron laser (FEL) beam composed of high-fluence X-ray bunches mere femtoseconds long [1,2]
Despite the intensity of the X-ray bunch, each is of such short duration that insufficient time is available for the changes associated with radiation damage to progress sufficiently before the diffracted X-rays have departed with their structural manifest to be recorded
Temperature phase (LCP), in which membrane proteins can be grown by the in meso method, might provide an alternative transport medium for SFX
Summary
Serial femtosecond X-ray crystallography (SFX) is a relatively new method for collecting crystallographic information on small crystals fed continuously into a free-electron laser (FEL) beam composed of high-fluence X-ray bunches mere femtoseconds long [1,2]. Temperature phase (LCP), in which membrane proteins can be grown by the in meso method, might provide an alternative transport medium for SFX. A lipid synthesis programme in the Membrane Structural and Functional Biology Group’s laboratory provides these MAGs in support of the in meso method of crystallization [19] Given their success, several are commercially available. Components of the precipitant stabilize a transition locally to the lamellar phase into which proteins diffuse to preferentially partition, concentrate and subsequently nucleate giving rise to macroscopic crystals [22] The latter are noted for generally tending to be small, but of high diffraction quality, and major effort is usually required to optimize conditions that produce crystals large enough for synchrotron radiation-based data collection. Attesting to the growing interest in the method, almost 60 new in meso records have been added to the PDB since the beginning of 2012
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