Since we were talking about GPCRs the other day, here's a nice overview of some of the experimental challenges associated with membrane proteins and how researchers are trying to overcome them. These challenges are associated not just with the crystallization, but with the whole shebang. Although many clever tricks have emerged, we have a long way to go, and at least a few of the tricks sound like brute trial and error.
To begin with, it's not that easy to get your expression system to produce ample amounts of protein. As indicated, you often need liters of cell culture to get a few milligrams of protein. The workhorse for production is still good old E. coli. E. coli does not always fold membrane proteins well, but it still beats other expression systems because of its cost and efficiency. Researchers have discovered several tricks to coax E. coli to make better protein. For instance it turns out that cold, nutrient poor conditions and slower-growing bacteria produce better folded and functional protein (although the exact reasons are probably not known, I suspect it has to do with thermodynamics and the binding of chaperones). Adding lipids from higher organisms to the medium also seems to sometimes help.
What’s more interesting are efforts to do away with cellular production altogether and just add reagents to cell lysates to jiggle the protein-production machinery. For some reason, wheat-germ lysates seem to work particularly well. There are companies willing to use these lysates to produce hundreds of milligrams of protein. One of the advantages of such cell-free systems is that you can add solubilizing agents and detergents to stabilize the proteins. A striking fact emerging from the article is how many private companies are engaged in developing such technology for membrane proteins; the end "credits" list at least a dozen corporate entities. The list should be encouraging to visionaries who see more fruitful academic-industrial collaborations in the future.
Then of course, there’s the all-important problem of crystallization. Of the 50,000 or so structures in the PDB, hardly a dozen are of membrane proteins. Membrane proteins present the classic paradox; keep them stable in the membrane and methods like crystallography and NMR cannot study them, but take them out of the membrane and, divorced from the protective effects of the lipid bilayer, they fall apart. Scientists have worked for years and come up with dozens of tricks to circumvent this catch-22. Adding the right kind of detergents can help. In the landmark structure of the beta-2 adrenergic receptor that was solved in 2007, the researchers used two tricks: attaching a stabilizing antibody to essentially clamp two transmembrane helices together, and replacing a disordered section of the protein with a T4 lysozyme, both strategies geared toward stabilizing the protein.
In the end though, there is really no general strategy and that’s still the cardinal bottleneck; as the article's title says, a "trillion tiny tweaks" are necessary to make your system work. What works for one specific membrane protein fails for another. As one of the pioneers in the field, Raymond Stevens from Scripps says, “People are always asking what the one strategy that worked is. The answer is there wasn’t one strategy, there were about fifteen”.
This is why chemistry (or economics) is not like physics. Although there are general rules, every specific case still invokes its own principles. In fields like membrane protein chemistry, it is unlikely that a single holy-grail strategy could be discovered that could work for all of them. The medley of techniques applied to membrane proteins makes the science seem sometimes like black magic and trial-and-error. All this makes chemistry hard, but also very interesting; if only a dozen membrane proteins have their structures solved, think of how many more are waiting in the shadows, awaiting the fruits of our sweat and toil.
Baker, M. (2010). Making membrane proteins for structures: a trillion tiny tweaks Nature Methods, 7 (6), 429-434 DOI: 10.1038/nmeth0610-429