Se-MAG Is a Convenient Additive for Experimental Phasing and Structure Determination of Membrane Proteins Crystallised by the Lipid Cubic Phase (In Meso) Method

Abstract

Both intensity and phase information are needed for structure determination by macromolecular X-ray crystallography. The diffraction experiment provides intensities. Phases must be accessed indirectly by molecular replacement, or by experimental phasing. A popular method for crystallising membrane proteins employs a lipid cubic mesophase (the in meso method). Monoolein is the most popular lipid for in meso crystallisation. Invariably, the lipid co-crystallises with the protein recapitulating the biomembrane from whence it came. We reasoned that such a lipid bearing a heavy atom could be used for experimental phasing. In this study, we replaced half the monoolein in the mesophase with a seleno-labelled analogue (Se-MAG), which has a selenium atom in the fatty acyl chain of the lipid. The lipid mixture formed the cubic mesophase and grew crystals by the in meso method of the alginate transporter, AlgE, and the lipoprotein N-acyltransferase, Lnt. Se-MAGs co-crystallised with both proteins and were used to obtain phases for high-resolution structure determination by the selenium single-wavelength anomalous diffraction method. The use of such a mixed lipid system may prove to be a general strategy for the experimental phasing part of crystallographic structure determination of membrane proteins that crystallise via the in meso method.

Keywords

• alginate transporter
• co-crystallisation
• experimental phasing
• lipoprotein <i>N</i>-acyltransferase
• selenium-sulfur hydrogen bond
• seleno-monoacylglyceride