In seeking out ideal conditions for growing protein crystals, solutions have increasingly been found in the low-gravity conditions of space. But answers might be lurking in fields closer to home.
Culturing high-quality protein crystals has, in the past decade, undergone a steady transformation from an art to science. That process has been assisted by exploiting the 'microgravity' conditions of space missions to lessen the fluid flows that disturb crystal growth on Earth's surface. As they describe in Applied Physics Letters, Heijna et al.1 use an alternative approach: very strong, but inhomogeneous magnetic fields with which they establish a tunable gravity environment that, for crystal growth, recreates space on Earth.
Protein crystals are highly sought-after commodities for many basic studies in biochemistry and structural biology, and for structure-based drug design. The better the quality of a crystal, the better the structural information it yields. Microgravity conditions reduce buoyancy-driven turbulent flows in the 'mother liquor' from which a crystal emerges, and so are thought to promote crystal nucleation and ideal growth. In addition, such conditions remove the sedimentation effect of crystals heavier than the mother liquor. These near-perfect conditions have indeed been used to deliver bigger and better-formed protein crystals, to perform fundamental studies of crystal quality, and to produce homogeneous distributions of crystal sizes2.
But experimentation in space has its disadvantages: restricted access, high costs (albeit mitigated by the small weight of the apparatus required) and political pressures, to name a few3. In addition, creating true microgravity conditions is difficult. Astronaut activity, for example, causes periods of gravity-like disturbance ('g-jitter')4. Although space has produced benchmark results, methods that are solely Earth-based have obvious attractions.
The inhomogeneous field (IHF) method harnessed by Heijna et al.1 exploits a vertical magnetic-field gradient to create a force that counterbalances gravity. This approach is the basis of magnetic levitation techniques that have been used, among other things, to make frogs hover5. The precise values to which the field and its gradient must be tuned to negate gravity depend on the nature of the crystals' mother liquor and its density. By creating effective gravity conditions from g (normal gravity) down to −0.15g (inverted gravity), the authors were able to slow down, halt and even reverse convection in the mother liquor (Fig. 1). An ingenious optical viewing set-up within the 32-mm-diameter borehole containing their magnetic field allowed them to view and monitor the growing crystal and its surrounding fluid directly.
This control of crystal-growth conditions is different from that brought about by microgravity: because the crystal and mother liquor respond to the magnetic field to different extents, convection (a property of the fluid) and sedimentation (a property of the crystals) are not eliminated simultaneously. This can be viewed in two ways. First, it is a limitation of the magnetic-field method. But second, it allows the experimental conditions 'convection-free' and 'sedimentation-free' to be separated out, and their relative importance in the growth of protein crystals to be evaluated. A caveat here is that, in an experiment to explore the accuracy of the settings in an IHF chamber used to grow inorganic crystals, residual fluid flows equivalent to around 0.5 µm s−1 — about the same level as g-jitter in space — are found even when gravity is perfectly balanced out6.
Besides the IHF approach, other methods, for example those using gels7 and microfluidics, can provide the advantages of microgravity on Earth. Microfluidics, when combined with robotics for accurate and systematic screening of growth conditions, allows crystal-growth droplets as small as 10−11 m3 (a hundredth of a cubic millimetre) to be accurately manipulated. In these small volumes, the problems of convection-driven fluid flows and sedimentation are scarcely relevant.
Of course, a crystal growing in such a small drop is also limited in size, but this presents little problem: modern synchrotron radiation facilities can analyse sample volumes of side just 20 µm (equivalent to about 10−14 m3). An upgrade programme under way at the European Synchrotron Radiation Facility in Grenoble, France, to narrow the focus of its probing X-ray beam will lower this limit still further. In the upcoming new world of crystals numbering just a few thousand unit cells — 1,000 cells being 10 by 10 by 10 units — beams focused to 0.1 µm or less, equivalent to a probed volume of 10−21 m3, will be required. Indeed, a challenge to the ingenuity of the engineers will be to incorporate the microfluidic and robotic stages necessary for the manipulation of such small volumes within the constrained volume of an IHF apparatus.
Protein crystallography with neutrons, which has the big advantage over X-rays of finding the positions of hydrogens (as deuterium atoms) even at relatively modest diffraction resolutions8, uses larger protein crystals. But even here, improvements in neutron apparatus and new protein deuteration facilities at the Laue Langevin Institute in Grenoble, and from 'megawatt spallation' neutron sources, will allow the reduction of sample sizes to just a fraction of a millimetre.
It is important to mention that it is not just inhomogeneous magnetic fields than can play their part in protein-crystallization techniques — the use of homogeneous magnetic fields was pioneered earlier by Sazaki and Ataka9,10. When crystals of the protein lysozyme are grown in a homogeneous magnetic field of 10 tesla, charged amino-acid residues in the structure showed increased order through the restraining effect of the magnetic 'Lorentz' force on moving electric charges11. These crystals remained stable when removed from the magnetic field for X-ray synchrotron analysis. Most recently, the use of combined magnetic and electric fields has permitted a quite remarkable homogeneity of protein-crystal size and shape12. The use of electric fields on their own in protein-crystal growth is well established13. A recent innovation here is an apparatus with multiple wells for crystal growth14.
The variety of these methods for optimizing crystal growth, as well as new synchrotron X-ray and neutron sources and the attractions of microfluidics and robotics, all add up to an exciting time in the analysis of biological crystal structures. Earth-based techniques such as those explored by Heijna et al.1 will ensure that researchers will keep, if not their imagination, then their equipment on the ground.