Protein Crystallization Hits

Detergents are an interesting class of molecules, mainly because of their amphipathic, or amphiphilic nature. They like both water & oil and are hence used extensively to keep hydrophobic molecular species dissolved in an aqueous environment. As a result they're used on an industrial scale to clean clothes, but they're also used in protein crystallization. Of course everybody thinks membrane proteins now, but that's not where this blog post is going.  Detergents can be used to optimize the crystallization of soluble proteins.

For example, in their 2001 paper Guan et al. describe the optimization of four soluble protein crystallizations with detergents, all resulting in improved crystal quality and crystal growth reproducibility.

Guan, R.-J., Wang, M., Liu, X.-Q, Wang, D.-C.
Optimization of soluble protein crystallization with detergents
Journal of Crystal Growth 231 (2001) 273-279

The detergents they use are rather diverse: they use Zwittergent, Mega-8, n-Octanoylsucrose, C12E8 and C-HEGA-10. The surprise here is that none of these detergents - with the exception of C12E8 - are routinely used in membrane protein crystallization trials.

Check out their nice images of protein crystals grown with and without detergent additives. 

These dirty crystals could use some detergent scrubbing…

Their simple detergent additive protein crystallization trials have provided the following outcomes: 

• polycrystalline -> single crystals
• crystal clusters -> single crystals
• poor X-ray diffraction-> 2.5A diffraction
• non-reproducible -> reproducible

Worth a try, I'd say.

Peter

There are so many parameters to change when optimizing crystallizations and they all need to be tried out to identify the critical parameter that improves X-ray diffraction. In this series of blog posts I've mostly discussed modulating the crystallization reaction, by adding additives, changing precipitant or protein concentration etc. However, there seems to be a simple way to optimize, keeping all other parameters constant: drop volume, temperature, all concentrations. What could it be?

Try switching the crystallization plate!

Jenny Martin describes in her recent PLOS paper with statistical rigour that different 96-well crystallization plates - formats, materials - do effect the outcome of protein crystallization experiments:

King, Gordon J., Kai-En Chen, Gautier Robin, Jade K. Forwood, Begoña Heras, Anil S. Thakur, Bostjan Kobe, Simon P. Blomberg, and Jennifer L. Martin.
Interaction between Plate Make and Protein in Protein Crystallisation Screening.
PLoS ONE 4, no. 11 (November 16, 2009): e7851

In fact, she suggests to optimize protein crystal growth by matching the protein with its optimal plate make. 

Figure: Compare different protein crystallization plates to grow optimized protein crystals.

So, here's a selection of 6 different Emerald BioSystems protein crystallization plates with different formats, well arrangements, volumes and materials that you may want to try to optimize protein crystal growth:

Clover 384 plate (COC material, 4 micro crystallization wells / reservoir)
Clover 384 plate (Polystyrene, 4 micro crystallization wells / reservoir)
Compact Clover plate (Polypropylene, 4 crystallization wells / reservoir)
Compact, Jr. plate (Polypropylene, 1 crystallization well / reservoir)
Combi Clover plate (Polypropylene, large wells, 4 crystallizations well / reservoir)
Combi Clover Jr. plate (Polypropylene, large wells, 1 crystallization well / reservoir)

Sometimes it's time to change the game.

Peter

This week I came across a message from PDBsum letting us know that certain figures and captions of a paper we had published in 2008 (Gerdts et al. (2008). Acta Crystallogr D Biol Crystallogr, 64, 1116-1122.) had been included into PDBsum. I had not visited the PDBsum site before and was at first intrigued and then positively surprised about the wealth of information that was presented on a protein structure (3cxk). This is a high-density, information rich way to get a quick impression on a protein structure and other accessory information.

Figure: Screenshot of the PDBsum entry for 3cxk. The crystallization experiment is nicely referenced in PDB-sum.

What I liked in particular about this presentation is that the crystallization experiment becomes part of the story. Our paper described an earlier, beta version of the MPCS - the plug-based nanovolume microcapillary protein crystallization system. Since the publication of the paper in 2008 the technology has matured substantially (check out the New Product Award 2010 that the PlugMaker has received last week).

Any context that goes beyond just reporting the precipitation reagent helps. Having such exquisite detail available when trying to reproduce protein crystallization experiments is often necessary to build on published research.

Way to go EMBL-EBI!
Peter