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Since its development in 1996 by Ehud Landau and Jurg Rosenbusch(1), Lipidic Cubic Phase (LCP) crystallization has led to the determination of over 300 membrane protein structures. In the past three years (2015-2017), LCP crystallization has been the method for over 30% of the 142 unique membrane protein structures determined by X-ray crystallography. Notably, nearly all of the unique GPCR protein structures determined during this time period have been crystallized using this method.


Although many novel host lipids have been developed for reconstitution into LCP(2, 3), Monoolein (9.9 MAG) still remains the most commonly used. In addition to the wide variety of host lipids, many additives have been used to alter the properties of the cubic phase, and make this method applicable to a wide family of membrane proteins(4, 5). Below, we will focus on two additives for LCP crystallization: Cholesterol and Distearoyl Phosphatidylglycerol (DSPG).
 
Cholesterol
The doping of cholesterol into host lipids has proven invaluable for the crystallization of GPCR proteins(6). The table below contains all of the unique structures from the past three years that have been reconstituted into a mixture of monoolein : cholesterol for LCP crystallization. The majority of these structures are GPCRs, except the Human sigma-1 receptor and the CD81 tetraspanin. Typically, the ratio of monoolein : cholesterol used to reconstitute these membrane proteins is 10:1. To prepare this mixture both the monoolein and cholesterol are dissolved in chloroform, which is then evaporated using an inert gas.


 

Year PDB Structure Reference
2017 5UEN A1 adenosine receptor (7)
2017 5XSZ LPA6 Lysophosphatidic Acid Receptor (8)
2017 5UNF Angiotensin type II receptor (9)
2016 5HK1 Human sigma-1 (σ1) receptor (10)
 
2016 5CXV
5DSG
M1 & M4 human muscarinic acetylcholine receptor (11)
2016 4ZJ8 OX1 orexin receptor (12)
2016 5L7D Smoothened (SMO) receptor (13)
2016 5TGZ CB1 cannabinoid receptor (14)
2016 5LWE Chemokine receptor CCR9 (15)
2016 5U09 CB1 cannabinoid receptor (16)
2016 5TCX CD81 full-length tetraspanin (17)
2015 4S0V OX2 orexin receptor (18)
2015 4XNV P2Y1 receptor (19)
2015 4YAY Angiotensin type I receptor (20)
2015 4Z34 LPA1 Lysophosphatidic Acid Receptor 1 (21)
Table 1: LCP Structures determined using monoolein doped with cholesterol as the host lipid.
 


DSPG
One disadvantage of monoolein as a host lipid is that membrane proteins with large intracellular or extracellular domains often cannot be reconstituted due to the small solvent channels of the cubic phase. In a recent paper published in Nature Communications(22), the lab of Raffaele Mezzenga from ETH Zurich characterized the anionic lipid additive distearoyl phosphatidylglycerol (DSPG) to swell the solvent channels of the host lipid Monopalmitolein (9.7 MAG). They used the monopalmitolein : DSPG mixture to reconstitute and crystallize the Gloeobacter violaceus ligand-gated ion channel, a protein which has previously been recalcitrant to crystallization using standard LCP techniques. Also of note, these LCP crystallization experiments were performed in the Laminex Plates available from Molecular Dimensions.
 
New Products from Anatrace and Molecular Dimensions for LCP crystallization

To save you time and simplify your workflow, we are excited to launch our new premixed monoolein : cholesterol for LCP crystallization (LCP18-CH200). This mixture contains a 10:1 ratio of monoolein : cholesterol and can be used directly for LCP experiments. No more dissolving lipids in chloroform and worrying if all of the solvent is removed!
 
This month, we are also adding DSPG (P716) to our growing catalog of lipids. Until May 31st, we are offering a 5% discount off your entire purchase when you include any pack size of our new monoolein : cholesterol mix (LCP18-CH200), our new DSPG lipid (P716), and our existing Monopalmitolein (LCP16) with your order (all three must be purchased). Use coupon code MarchLCP online or contact Customer Service for details on how to take advantage of this offer!
 
Lastly, Molecular Dimensions is launching MemGoldMeso, a new screen for the crystallization of membrane proteins using LCP. This screen is the result of a survey of all structures deposited in the PDB and solved using the mesophase method up to March, 2017. This analysis was performed by the developers of the popular MemGold and MemGold2 screens, Prof. Simon Newstead and Dr. Joanna Parker of Oxford University.

 

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. Ishchenko, A., et al. (2017) Cryst Growth Des. 17(6), 3502-3511.
  4. Cherezov, V., et al. (2002) Biophys J. 83(6), 3393-3407.
  5. Cherezov, V., et al. (2006) J Mol Biol. 357(5), 1605-1618.
  6. Caffrey, M., et al. (2012) Biochemistry 51(32), 6266-6288.
  7. Glukhova, A., et al. (2017) Cell 168(5), 867-877.
  8. Taniguchi, R., et al. (2017) Nature 548(7667), 356-360.
  9. Zhang, H., et al. (2017) Nature 544(7650), 327-332.
  10. Schmidt, H. R., et al. (2016) Nature 532(7600), 527-530.
  11. Thal, D. M., et al. (2016) Nature 531(7594), 335-340.
  12. Yin, J., et al. (2016) Nat Struct Mol Biol. 23(4), 293-299.
  13. Byrne, E. F. X., et al. (2016) Nature 535(7613), 517-522.
  14. Hua, T., et al. (2016) Cell 167(3), 750-762.
  15. Oswald, C., et al. (2016) Nature 540(7633), 462-465.
  16. Shao, Z., et al. (2016) Nature Nov 16. doi: 10.1038/nature20613. [Epub ahead of print].
  17. Zimmerman, B., et al. (2016) Nature 167(4), 1041-1051.
  18. Yin, J., et al. (2015) Nature 519(7542), 247-250.
  19. Zhang, D., et al. (2015) Nature 520(7547), 317-321.
  20. Zhang, H., et al. (2015) Cell 161(4), 833-844.
  21. Chrencik, J. E., et al. (2015) Cell 161(7), 1633-1643.
  22. Zabara, A., et al. (2018) Nat Commun. 9(1), 544.