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 A simple approach that can reshuffle the ingredients from several crystallization hits

Imagine that you've run some screens and found the crystallization hits shown on the left. Now, somehow, you want to combine the ingredients of those three hits to get something like the crystal shown on the right.

We've introduced a new script for the Oryx range of robots that can optimize crystallization by reshuffling the ingredients from several hits.  (It's useful for seeding and ligand-binding experiments too - see below).

Say you've picked up needles or small crystals in the following conditions: 

1.  30 % v/v PEG4000, 0.2M MgCl2, 0.1M TRIS, pH 8.5 

2.  28 % v/v PEG400, 0.2M CaCl2, 0.1M HEPES, pH 7.5 

3.  1.0M Na acetate, 0.1M imidazole, pH 6.5 

Looking at these results, it's hard to draw up a plan for optimization.  Is the best precipitant PEG, or a salt?  If PEG, does the molecular weight matter?  Are the bivalent cations Mg2+ and Ca2+ important?  And which pH is best? 

Our new script allows the precipitants to be dealt with separately from the other ingredients so that they can be reassorted.  For example the best crystals might come from the high-lit ingredients above.

Over the years we've heard how successful "targeted" screens can be.  The trouble was that they were time-consuming to set up, and - until now - separate robots were needed to make the screen and to set up the drops. The new Combinatorial Optimization script is based on a simple idea, but it allows a (systematic-type) targeted screen to be set up in 15 to 20 minutes. 

 The optimization of microseeding

Note that the same approach can be used for several other important experiments. For example, microseeding often gives too many crystals. Using the script, seed stocks with various dilutions can be added to one or more hit conditions. (Typically a dilution series would be set up, running from the "neat" seed stock to a 1:100,000 dilution.) The crystals below were optimized by Laura Cendron using a similar approach. 

Crystals grown by Laura Cendron at the University of Padova. Left, crystals used to make the seed stock. Other images: crystals grown with seeding.

Other applications

The same script can be used for additive experiments.  For example, up to 12 ligands or inhibitors can be added to the twelve rows of the plate. 

To request a demonstration in your own lab, please contact Hilary@douglas.co.uk.

Collecting data at Diamond Light Source?  We're located about 20 minutes from Diamond, and we'd be happy to pick you up and demonstrate our systems in our lab.  If you take some protein samples with you, we can also set up some crystallization trials for you, including microseeding and combinatorial optimization.

Douglas Instruments

Douglas Instruments was founded in 1987 by Peter Baldock and Patrick Shaw Stewart. In 1989, in collaboration with David Blow’s group at Imperial College (London), Douglas Instruments developed their first system for automatic protein crystallization – the IMPAX. The company moved in 1999 to Douglas House, about 25 miles South of Oxford, where its Oryx range of protein crystallization systems has been developed.

Over 200 crystallization units have been installed since 1987 in universities, research institutes and companies worldwide. Oryx systems offer versatile solutions for protein crystallization and Douglas Instruments continues to work closely with its customers to develop novel scientific techniques in this area. We are always happy to hear from workers in the field, whether they are customers or not. If you have any comments, questions or suggestions please e-mail Hilary@douglas.co.uk or Patrick@douglas.co.uk .

Here are some of the meetings in 2012 where we will be demonstrating the Oryx: 

Seventh International Workshop on X-ray Radiation Damage to Biological Crystalline Samples, Diamond Light Source, 14 - 16 March 2012.

BCA Spring Meeting, University of Warwick, 16 - 19 April 2012

FEBS Advanced Methods in Macromolecular Crystallization, Czech Republic June 22 - 29, 2012

ACA Boston 28 - 31 July 2012

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