Once you've got a hit with your membrane protein crystallization trial, your life may get really exciting. After confirming that the hit is actually a protein crystal and obtaining even the weakest X-ray diffraction patterns, you're getting into the optimization game. One of the obvious parameter to optimize is the crystallization cocktail. How can that be done without wasting your precious protein sample on unproductive crystallization conditions?
Designing and making grid-screens around a particular hit condition is complicated due to the presence of detergent in the crystallization experiment. An established approach is to be close to the detergent phase separation boundary while avoiding the 'heartland' of phase separation. How can this be done practically? You could check out the various papers on this topic and spend a lot of time searching and possibly finding hints on formulations that are relevant to your hit. You could also search the phase boundary conditions in a pre-screen type of experiment (a la Song & Gouaux: Membrane protein crystallization: application of sparse-matrices to the alpha-hemolysin heptamer Methods in Enzymology(1997) (60-73)). The quickest way though to obtain guidance on optimization screen design is through mining of existing detergent phase boundary data. Fortunately Mary Koszelak-Rosenblum et al. from HWI have made such data mining a quick and simple experience:
Koszelak-Rosenblum M, Krol A, Mozumdar N, Wunsch K, Ferin A, Cook E, Veatch CK, Nagel R, Luft JR, Detitta GT, & Malkowski MG (2009). Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins. Protein science : a publication of the Protein Society, 18 (9), 1828-39 PMID: 19554626
The data comes in an Excel spreadsheet called SLICKSPOT. This tool is available for download here.
The input parameters for this nifty tool are type and concentrations of:
- Detergent (data is available for C10M, C12M, b-HG, b-OG, b-NG, CHAPS, LDAO, C12E8, C8E4, C8E5, FC-12),
- crowding agent PEG (data is available for: PEG400, PEG1000, PEG2000, PEG2000MME, PEG3350, PEG 4000, PEG5000ME, PEG6000, PEG8000, PEG20000)
- Salt (data is available for CaCl2, KCl, LiCl, Li2So4, MgCl2, Na2C3H2O4, NaCl, NaH2PO4, (NH4)2SO4, NH4H2PO4, (NH4)2HPO4)
So, let's say the membrane protein crystal hit was produced at room temperature with a formulation that contained 1% Octylglucoside, Ammonium sulfate and PEG4K. For these conditions Slickspot produces the following output as a chart:
Example of a Slickspot output. How to read this customized chart: Salt and PEG concentrations below the blue curve form a single phase, those above are phase separated. Sticking to conditions around the blue line is preferred.
So, what's to do from here?
Design your customized formulation screen with Ammonium sulfate and PEG4000 conditions near to the blue line.
Pretty slick, hm?
Peter
PS: I just realized that Slickspot fails to update the legend when additional conditions are queried; a cosmetics-only bug I hope.