Dry solutions for crystallography

To some, X-ray crystallography may be a rather dry topic. However, the process generally entails lots of liquids. In particular, the commonly used practice of crystal soaking entails transferring protein crystals to a new solution containing dissolved ligands, which is both tedious and can cause crystals to shatter or dissolve. A new paper by Jean-Francois Guiçhou at Université de Montpellier and collaborators in Acta Cryst. D aims to streamline the process, and so lower barriers for obtaining structural information that could guide drug design.

Rather than manually transferring crystals to new solutions, the researchers pre-coated crystallization plates with ligands and then grew protein crystals in them. They first dissolved the ligands, transferred these to the wells, and allowed the solvent to evaporate. Although they tested a variety of solvents, including acetone, tetrahydrofuran, ethanol, acetonitrile, 2-propanol, water, and DMSO, only the last two proved suitable; most of the rest wicked up the well, spreading over too large of a surface (though methanol has been used by Beryllium, née Emerald). DMSO is, of course, the most commonly used solvent for storing small molecules, and so should work for most ligands. DMSO is not very volatile, but only 1 µl was used per well, and putting the plates in a fume hood for a week left behind dry ligand.

To make things easier still, the researchers used special crystallization plates that could be put directly into an X-ray beam (in situ crystallography), further diminishing the amount of manipulation required. The technique was tested against four different proteins: the old standard hen egg-white lysozyme and the drug targets cyclophilin D, PPARγ, and Erk-2.

For lysozyme, the water-soluble fragment benzamidine was used, and the resulting structures showed the fragment binding in a similar manner as previously described. So did structures of PPARγ bound with the high affinity ligand rosiglitazone. Cyclophilin, though, was not as successful: of nine fragments attempted, only one produced a structure. In contrast, three fragments produced structures using conventional approaches. ‘Dry’ crystallization was more successful with two more potent (micromolar or better) cyclophilin ligands. Interestingly, dry crystallization succeeded with one ligand that had previously been characterized only by co-crystallization; even week-long soaking experiments had not worked.

Finally, Erk-2 was screened against 14 ligands designed as hinge-binders with low solubility in water. Crystals were obtained with five of the ligands, and four were large enough to generate good-quality structures.

Overall this seems like a convenient approach, though it does seem prone to false negatives. What do the crystallographers out there think – is this a practical solution?