Protein Crystallization Hits

How long does it take to set up a protein crystallization experiment?

It all depends on the equipment and the state of organization in your lab. Even with modest manual dexterity, setting up a single 96-well tray really shouldn't take more than 10 min. Allow 10 min of preparation time and you're at only 20 min. In fact, without the use of robotics it is possible to process a 40 uL protein sample and prepare a 96-well protein crystallization trial within less than 20 minutes, all things considered. Multiply that by 2 or 4 if you aim to increase coverage of crystallization phase space. Here's a rough schedule for preparing a single tray consisting of 96 x 0.8 uL sitting drop protein crystallization experiments using the vapor diffusion method :

Is setting up a single crystallization tray taking you more than 20 min? Maybe it's worthwhile getting those multichannel pipettors and repeating dispensers, or switching from slow hanging drops to fast sitting drops.

Or just clean up the lab and get those utensils that you already have, better organized ? ;)

Cheers, Peter

There's a lot of lab folklore going around in protein crystallization labs. These are stories such as: "the only thing that every worked for my protein XY is this odd trick" followed by a description of an exotic crystallization trick. Understood, often it is difficult to reconcile what exactly is going on in a particular crystallization experiment. Sometimes, however there's evidence that seemingly weird crystallization tricks do work - check out this example, where seaweed made the difference. As much as we like to get experimentally tested evidence, in general this rule should hold: 'who crystallizes is right'.

Along these lines, Ivana Tomcova and Ivana Smatanova have come across an interesting case of crystallization dependence that they call 'cross-crystallization'. In their paper

CROSS-CRYSTALLIZATION AS A NEW OPTIMIZATION TOOL OF CRYSTALLIZATION PROCEDURES
Materials Structure, vol. 14, no. 1 (2007)

they describe the crystallization of cytochrome c4 from anaerobic purple sulphur bacterium Thiocapsa roseopersicina. They used the Emerald BioSystems CombiClover Crystallization plate
(thank you very much!) in a special way: all crystallization chambers were filled with a different additive (chloride salts of copper, cadmium, cobalt and barium) and protein was only added to the cupric chloride containing chamber. They optimized this recipe (5 mM CuCl2, ammonium sulfate, citric acid buffer pH5) to a point where the neighboring crystallization chambers were required to contain metal salts (CdCl2, BaCl2, CoCl2), otherwise crystals would not show up. 

Fig: Clover Crystallization Plates

There's not much to say other than: apparently it works.

Cheers,

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