by Peter Nollert
November 9, 2010 22:54
Handling and storage of protein crystallization trials is usually carried out in a manner that minimizes exposure to vibration. I have set up crystallization trials and put them aside to a 'safe place' with the intention to let the crystallization commence without me interrupting the crystallization process. While I'm not sure if it was the lack of shaking or the temperature increase during observation caused by the microscope light, I did get larger and better diffracting crystals in at least one case. On the other hand, I've seen crystallization cabinets with hundreds of trays that got a whack every time somebody did not catch the door into the crystallization room. And this was a very productive protein structure lab.
Let's use this vibration effect and take it to the extreme: how about adding a bit more stress, such as ultrasonication? 3 seconds of of bath-ultrasonication every 3 minutes throughout the crystallization process?
That's exactly what Crespo et al have done and describe in their new paper:
Crespo, R., Martins, P., Gales, L., Rocha, F., & Damas, A. (2010). Potential use of ultrasound to promote protein crystallization Journal of Applied Crystallography, 43 (6) DOI: 10.1107/S0021889810040951
This is the contraption they used to compare with ultrasonication vs. without.
Figure: Simple setup to compare the effect of micro batch-type protein crystallization with ultrasonication and without ultrasonication.
The setup is rather well controlled and was used to map the supersaturation curves for Lysozyme (I know…) in the presence and absence of the ultrasonication stress regime. The finding is that ultrasonication promotes nucleation. In doing so, it appears that crystals gown with this method are of higher quality. While the statistics on crystal quality (n=13) are not that convincing and the effect is small (0.1A average improvement), I'd do it if I'm desperate. For instance in cases where protein crystallization optimization by
have not yielded any improvements in the protein crystal diffraction quality.
Cheers,
Peter
by Peter Nollert
March 16, 2010 15:00
by Peter Nollert
December 24, 2009 15:00
The end of 2009 is near and it's time to clean up to make room for new and exciting projects in 2010. I'm just returning from the lab with a stack of protein crystallization trays to toss. Some of the crystallization experiments were prepared more than a year ago! There were two categories of crystallization trays that I dealt with:
1. Trays set up with protein that have never yielded any crystals at all
Should I keep them and hope that via slow desiccation or proteolytic cleavage protein crystals will eventually form? Nah! Everything that's older than 6 months must go. Gone they are. - I'm having second thoughts though, now that I'm writing this. I could open the crystallization chambers for a while and let the drops dry out just a little and then close them again. After all, protein crystallization by dehydration does work sometimes. Or move the trials to a different temperature? Or add chemotrypsin to the drops and create target fragments that crystallize.
Crystallizers' remorse setting in big time....
2. Trays set up with protein that have yielded crystals and structures
I could throw them out altogether. Crystals diffracted, structure is done. But, why not keep a few trays with crystals? You never know when a project 'comes back' - with the need to co-crystallize together with a small molecule ligand or protein partner. Even if the crystals don't diffract they may be useful and serve as seeds.
Pretty (Ammonium sulfate) crystals. These are the easy protein crystallization setups to give up on and throw out.
Phew! - that was easy, actually.
2010 here I come!
Peter
by Peter Nollert
December 2, 2009 04:08
One of the general advice for storage of crystallization experiments is to store them 'in the dark' or in 'dim light'.
Since I started my crystallization career with light-sensitive proteins this made a lot of sense to me. I'm wondering though, if the advise also holds up for the crystallization of light in-sensitive protein samples.
Turns out that at least some form of light can alter the outcome of crystallization experiments. Let's look at light that gets absorbed by protein, mostly via the aromatic amino acids Tryptophan, Tyrosine and Phenylalanine. These residues absorb light in the range of 280 nm, which can give rise to photo-oxidation reactions. Tstsuo Okutsu has published on the topic of UV light effect on nucleation.
Okutsu, T. (2007) hotochemically-induced crystallization of protein
Journal of Photochemistry and Photobiology, 8(3), 143-155
These experiments were carried out by 'weak UV irradiation' and there really shouldn't be a need to protect one's protein crystallization experiments from UV light because (i) there's little UV light around in a typical lab setting and (ii) most crystallization containers have poor UV transmission. Illuminating your samples with a strong UV source (i.e. UV microscope) is different though, and I'd like to hear from people what their experience is in this respect.
So, what about all the other light? Very strong pulses of light can apparently generate productive protein crystal nuclei. Check out this report where the effect of femto-second laser light on the formation of crystals of the membrane protein AcrB, ADA (adenosine deaminase) and human triosephosphate isomerase (TIM) was investigated. AcrB crystallization was dramatically improved. An ordinary laser pointer won't do the job, unfortunately.
Advanced protein crystal nucleation tool.
Finally, some of the formulations that are used in sparse matrix crystallization screening experiments are light sensitive and react over time. Hence the advice to store solutions containing Imidazol, MES buffer and Ammonium Iodide in dark containers. Once the photochemistry sets in, things become rather messy. That's not to say this is bad, since aggregated protein may serve as a nucleant. But light exposure is a factor in protein crystallization that should be carefully controlled.
Myth not busted.
Peter
by Peter Nollert
October 21, 2009 02:45
I deeply appreciate admitting mistakes. This is how we learn. Try, fail, try again with new spin and succeed. More power to crystallizers like Miriam L. Sharpe, who manages to get a paper out of a perceived failure. She didn't get any crystals in her initial "....attempts at crystallizing the protein, including screening 681 different conditions, were unsuccessful. Initial screens included Crystal Screens I and II (Hampton Research), a systematic PEG-pH screen (Kingston et al., 1994 ), a PEG/Ion screen (Hampton Research), Footprint Screen No. 1 and the PEG Footprint Screen...". Check out her complete story here.
So much for that. But guess what - Miriam sets up an entire tray with 100 nl protein drops each, and admittedly by mistake forgets to add any precipitant solution (neither in the well, nor in the drop). And what happens? Get this: seven months later she finds crystals in the dehydrated drops. She then goes on, dilutes the viscous matrix around the crystals, fishes them out before they dissolve, flash freezes, collects X-ray diffraction datasets and determines the 2.1A structure.
Crystals grown by incubation under 'carefully adjusted' dehydrating conditions, or: forgetting to add precipitation reagent and letting the crystallization tray sit on the shelf for 7 months.
Congratulations to the structure of Hupoxic response protein I, Miriam Sharpe! And thank you so much for this great crystallization story!
Peter
Crystals grown by incubation under 'carefully adjusted' dehydrating conditions, or: forgetting to add precipitation reagent and letting the crystallization tray sit on the shelf for 7 months.
Congratulations to the structure of Hupoxic response protein I, Miriam Sharpe! And thank you so much for this great crystallization story!
Peter