Standards in membrane protein structural biology
were high. With 7.7 MAG, they're even higher.

Since being first described in 1996(1), the Lipidic Cubic Phase (LCP) or in meso crystallization method has been used to determine the structures of many classes of membrane proteins, including GPCRs, transporters, enzymes, and channels, among others(2). In the past five years (2014-2018) there have been over 400 unique membrane protein structures determined by various methods, with nearly 20% of those structures being determined by LCP crystallization. The cubic phase is spontaneously formed by mixing a protein solution (or water) with a host lipid(3). The most commonly used host lipid for LCP crystallization is monoolein (9:9 MAG); however, many different host lipids and additives can be used, each having different properties(4).
 
One host lipid that has been successful in the crystallization of a number of challenging membrane proteins has been 7.7 MAG, which was first characterized by the lab of Martin Caffrey in 2004(5). The cubic phase formed by 7.7 MAG has very different properties compared to monoolein with a bilayer thickness which is 6.5 Å less (7.7 MAG: 25.8 Å, 9.9 MAG: 32.3 Å), and a water channel diameter that is 21.8 Å bigger (7.7 MAG: 61.5 Å, 9.9 MAG: 39.7 Å)(5). The larger water channel diameter is hypothesized to support crystallization of membrane proteins with larger intra or extracellular domains.

Perhaps the most well-known example of crystallization using 7.7 MAG as the host lipid is the 2011 structure of the β2 Adrenergic receptor-Gs protein complex published by Brian Kobilka and coworkers(6). In this example, monoolein was unsuccessful in crystallizing the complex, most likely due to the large intracellular components, including the hetero-trimeric Gs protein, T4 lysozyme, and the nanobody NB35, not being able to reside within the smaller water channel(7). Some additional structures that have used 7.7 MAG as a host lipid include:

 
  • 2011: The peptide antibiotic gramicidin D(8)
  • 2012: The caa3-type cytochrome oxidase(9)
  • 2014: β2 Adrenergic receptor in complex with nanobody Nb6B9(10)
  • 2016: The sodium protein antiporter NapA(11)

A growing collection of tools for LCP crystallization from Anatrace and Molecular Dimensions

We are excited to launch 7.7 MAG, the latest addition to the growing panel of LCP host lipids offered by Anatrace. Other host lipids include 9.9 MAG (monoolein), our premixed 10:1 monoolein:cholesterol, and 9.7 MAG (monopalmitolein). In addition to these host lipids, Molecular Dimensions offers a number of plates and screens for LCP crystallization, including:

 
  • Laminex Plates: A simple way to setup, visualize, and harvest crystals from LCP experiments.
  • MemGoldMeso: Our most up-to-date screen for LCP crystallization (developed by Simon Newstead and Joanne Parker at Oxford University), which contains conditions less biased towards GPCR and rhodopsin proteins.
  • MemMeso: Our original LCP crystallization screen developed by Osamu Nureki at the University of Tokyo.
 
References:
  1. Landau, E. M. and Rosenbusch, J. P. (1996) Proc Natl Acad Sci U S A 93(25), 14532-14535.
  2. Caffrey, M. (2015) Acta Crystallogr. F Struct Biol Commun. 71(Pt 1), 3-18.
  3. Caffrey, M and Cherezov, V. (2009) Nat Protoc. 4(5), 706-731.
  4. Li, D, et al. (2011) Cryst Growth Des. 11(2), 530-537.
  5. Misquitta, L, et al. (2004) Structure. 12, 2113-2124.
  6. Rasmussen, SG, et al. (2011) Nature. 477(7366), 549-555.
  7. Caffrey, M, et al. (2012) Biochemistry. 51(32), 6266-6288.
  8. Hofer, N, et al. (2011) Cryst Growth Des. 11(4), 1182-1192.
  9. Lyons, JA, et al. (2012) Nature. 487(7408), 514-518.
  10. Ring, AM, et al. (2013) Nature. 502(7472), 575-579.
  11. Coincon, M, et al. (2016) Nat Struct Mol Biol. 23(3), 248-255.