Test of the Granada Crystallization Box in Space

Orbital vehicles such us the Shuttle or the International Space Station offer the possibility of performing experiments under conditions of reduced gravity, for example crystallisation experiments in which the effects of sedimentation and the movement of fluids by convection are substantially reduced. Due to various perturbations, however, these effects are not completely avoided. Numerical simulations and experimental observations suggest that the utilisation of small capillary volumes (up to 50 ?L) by using small diameter capillaries (up to 1.0 mm) might totally eliminate both effects, and thus enable us to grow crystals in space in purely diffusive surroundings.

When crystals of biological macromolecules are sought, only very small quantities are usually obtained, due to the difficulty of extracting and purifying such macromolecules. Moreover, once the crystals have been obtained they must be transferred to a quartz capillary for X-ray diffraction analysis or to freeze the crystals in order to solve the X-ray structure.

The Granada Crystallisation Box (GCB) has been designed in the Laboratorio de Estudios Cristalográficos (LEC) at the CSIC (Consejo Superior de Investigaciones Científicas), on the basis of these considerations. The GCB drastically reduces the amount of protein required, enables multiple experiments to be performed within a small volume and reduces the perturbing effects of gravity. Moreover, the crystals form within an X-ray capillary, ready for diffraction with no further manipulation.

The GCB is under patent by the CSIC and licensed to NewBiotechnic, andalusian company developping new crystallisation products. GCB is commercialised by Hampton Research

GCB Operating Principles

The GCB consists of a small polystyrene box fitted with a guide channel, into which up to 6 glass capillaries can be placed. The bottom of the box is covered with a layer of gel, where the capillaries are inserted to a depth of about one centimetre. Then a solution of the precipitating agent, the substance that will provoke the crystallisation of the protein, is poured onto the gel.

Both before the launch of the orbital vehicle and during the flight until it goes into orbit, the precipitating agent diffuses through the pores of the gel. The insertion depth is calculated such that the precipitating agent reaches the bottom of the capillary when the GCB is orbiting the Earth in free fall, and so diffuses through the aqueous protein solution in the capillary. Under these circumstances, neither convection nor sedimentation perturb crystal growth.

The GCB functions by means of a counter-diffusion technique. This crystallisation method, tested by video-interferometry and microscopy in our experiments on the STS-95 mission, is based on coupling the diffusion of the precipitating agent through the protein solution with the precipitation of the crystal. This coupling creates a wave of supersaturation travelling across the capillary. The amplitude of the wave diminishes while its width increases. Thus, as the wave advances, the protein is crystallised under varying conditions of crystallisation, progressively approaching optimum conditions. This scanning process, which the GCB performs in a single experiment, is normally carried out in standard crystallisation techniques by means of multiple trial and error experiments.

Aims of the Experiment

The GCBs are designed to be used on ground (http://lec.ugr.es/). For experiments in space, a container must be prepared with specific materials, capable of withstanding the launch and re-entry, and that meets the safety requirements of the International Space Station (ISS). Furthermore, the container must prevent the escape of liquids or gases from the GCB. Twenty three GCBs (that is, 138 capillaries) will be placed in an aluminium container designed by NTE; this container has internal dimensions of 12 x 12 x 8 cm. The total weight of the experiment is 1.02 kg.

The GCB flight test will be realised on the occasion of the Andromede mission, a Franco-Russian taxi-flight project. The launch has been scheduled for August 21 from the Russian cosmodrome in Baikonour (Kazajstan).

The main objective of our experiment is to validate the concept and functioning of this apparatus, specifically designed for space. Each of the GCBs will contain a single protein, with its precipitating agent and additives. Six capillaries, with diameters ranging from 0.2 to 1.0 mm, will be placed inside each GCB. The results will be compared with those of identical experiments carried out on Earth, and with those of numerical simulations of fluid dynamics within capillaries, performed in conjunction with the MARS centre in Naples. Different proteins will be used, selected by LEC from proposals made by European and Russian laboratories. Records of humidity and temperature within the GCB will be obtained by means of custom-made devices.

Post - Flight Activities

After the flight, the position of the crystals inside the capillaries will be studied, noting whether these are influenced by capillary diameter, and comparing them with the experiments performed on Earth. We will study the stability of the capillaries in the gel and analyse temperature variations.

Crystal quality will be evaluated by X-ray diffraction within the same capillaries that the crystals grew in. The laboratory proposing and supplying each particular protein will carry out this evaluation, although the raw data will be shared with the LEC for comparative studies.

Preliminary Results

Although a complete analysis of the data obtained from the experiments of crystallization with the GCF in this mission is still in process, some preliminary results were presented in the 9th International Conference on the Crystallization of Biological Macromolecules, held in Jena, Germany in February 2002. The PowerPoint presentation discussed there is now available, also as HTML pages.