Protein Crystallization Hits

Sorting through my past years' crystallization reports I'm particularly glad about the fact that all of the crystal images are associated with a description of the crystallization precipitation cocktail. In the same image. Of course one can always go back to the original notebook and dig out the particular crystallization condition used, but time tends to cast doubts and having the well solutions directly associated with the crystals shown in  an image is very practical. Here's a simple way to pull out primary screen parameters for association with a particular crystallization experiment:

Let's say the crystallization was done with the JCSG+ screen, formulation from well E4

• Go to E-Screen Builder 
• Select Vendor (Emerald BioSystems),  pick screen: JCSG+
• Click on E4 - et voila, E4 contains 1260 mM Ammonium sulfate, 200 mM Lithium sulfate and 100 mM Tris at pH 8.5.
• From here you can take a screenshot, or use the 'Snipping Tool' to copy and paste this image right next to the image of the crystal in your report.

Finding a particular protein crystallization screen condition is simple: 1. select vendor, 2. select screen, 3. select hit. The crystallization formulation is displayed for you to (4) cut and paste into a crystallization report.

Easy, isn't it?

Peter

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.

Earlier this month I received an email from John Wiley & Sons, confirming that 'The supplement file is now available at http://onlinelibrary.wiley.com/doi/10.1110/ps.073263108/suppinfo'. More than half a year ago I had pointed out that the 'supplementary material' to a paper was not available online. Now it is. Took a while, but  THANKS A LOT for following through, John Wiley & Sons!

When you go to this page, you can download Simon Newstead's alpha-MP-database.xls.

Although somewhat outdated, this spreadsheet contains a concise summary of crystallization conditions from 121 alpha helical membrane protein structures that are listed in the PDB. The extensive analysis of crystallization conditions is described in the accompanying paper:

Newstead S, Ferrandon S, & Iwata S (2008).

Rationalizing alpha-helical membrane protein crystallization.

Protein science, 17 (3), 466-72

PMID: 18218713

While this accounting exercise is not the sexiest of all science, it is of great interest to all of us membrane protein crystallizers . The database is a handy tool that can be consulted when only small quantities of membrane protein sample is available and one is forced to focus on 'what's worked in the past'.  And of course, this data summary is incredibly for the design of new (although highly biased) crystallization matrices. This is exactly what Simon has done: based on his own analysis, a new sparse matrix crystallization screen, called MemGold  is described.

Great work!

Peter

This is the list of answers that I got today from a quick and informal survey that I conducted over 'lunch' with some of our crystallographers: 'what's been the best thing that happened in 2010 in the world of crystallization / crystallography?'

Here we go with the highly opinionated 'Best of 2010 in Protein Crystallography' (in no particular order):

• Iodide and Sulphur SAD phasing catching on big time
• Remote access to GM/CA-CAT & LS-CAT at the Argonne Advanced Photon Source
• Emerald's Plugmaker winning the 2010 Lab Automation New Product Award
• Fabrice Gorrec's Morpheus crystallization screen (it is a 2009 publication, but its impact was felt mostly in 2010)
• The entire 2010 April issue of Acta D on phasing, software tools and best practices

These are my two personal favorites in the 'Best New Software' category: Mendeley (keeping track of ones research PDF collection) and new de-novo feature of the E-Wizard online crystallization screen generator. But where's the 'best 2010 protein crystallization iPhone app?'.

Not directly related to the field of protein crystallography, but this one could serve as an example for us crystallographers:

• The clever choice of the name for the bacteria that have phosphate exchanged by arsenate atoms. Strain GFAJ-1 stands for "Give Felisa A Job" (first author of the paper is Felisa Wolfe-Simon).

Happy 'last days of the decade without a name',

Peter

This is my list of indispensable online tools that I would encourage every protein crystallizer to use during different phases of the protein crystallization endeavour: 

While there's some overlap with Sean's list over at P212121.com, he has a lot of additional tools listed that relate to up- and downstream crystallization processes. Sean's blog is definitely worth a visit!

All the best,

Peter