France: Producing large protein crystals and polycrystalline arrays using high magnetic fields

An aligned polycrystalline array suspended in a fixed hydrogel following alignment in liquid form while in the field.

An aligned polycrystalline array suspended in a fixed hydrogel following alignment in liquid form while in the field.

Growing large crystals (>0.1mm3) of biological macromolecules that are suitable for neutron crystallography is not an easy task. The solution to this problem holds the key to overcoming the single biggest difficulty that limits the technique, and would have a major impact on the range of biological problems that could be addressed using neutron protein crystallography.

Whereas it is often very difficult to grow large single crystals, it is typically far less of a problem to produce “showers” of crystals – immense numbers of very small crystals (microcrystals). In the pursuit of the growth of large single crystals, these showers are often regarded as a rather negative outcome.

However – what if there was a way to arrange many microcrystals so that they behave as if they were a large crystal?

The vial and holder that are placed in the magnet.

The vial and holder that are placed in the magnet.

Ashley Jordan from ILL, in collaboration with Elizabeth Blackburn (University of Birmingham (UK), now Lund University in Sweden) have been investigating how high magnetic fields can be used to align thousands of microcrystals in arrays such that useful crystallographic information can be extracted and used. This obviates the need for the growth of large single crystals and could in principle open up the field to a wide range of new problems.

The basis of this approach is to suspend the microcrystals in a hydrogel medium at a temperature above its melting point – so that the crystallites are free to move in the liquid. The sample is then placed within a strong magnetic field (17 Tesla – see picture below). Over the course of a day, the microcrystals undergo bi-axial alignment and orientate themselves along the field direction. When the temperature is subsequently decreased, the gel solidifies, fixing the microcrystals in their aligned position. For the purposes of diffraction, this makes the sample equivalent to a large single crystal. The fixed sample can then be removed from the field and brought to a diffractometer for crystallographic data collection.

17 Tesla magnet from the University of Birmingham, located in the guide hall at the ILL

17 Tesla magnet from the University of Birmingham, located in the guide hall at the ILL

This technique has been tested and developed using a number of model proteins including rubredoxin, trypsin, and lysozyme – with some quite striking results. Preliminary neutron diffraction experiments on the aligned microcrystals have been performed which show clear evidence of sample ordering. For these diffraction studies, experiments using a deuterated hydrogel gel/buffer have helped reduce the hydrogen incoherent scattering background. There is also clear evidence that the magnetic field influences the morphology and growth of the individual microcrystals and that these grow considerably larger in the presence of the field.

Acknowledgements: Trevor Forsyth and Ashley Jordan, ILL