The Granada Crystallization Facility (GCF) is a multiuser facility designed to conduct crystallization experiments of biological macromolecules in microgravity using a counterdiffusion technique inside capillaries. The counterdiffusion technique is implemented in the Granada Crystallization Boxes (GCBs).Facility Manager(s)
Triana Science and Technology, Granada, , Spain
NTE, Barcelona, , Spain
European Space Agency (ESA)Expeditions Assigned
3,5,6,7,8,9,10,12,14,15,16,17Previous ISS Missions
The Grenada Crystallization Facility (GCF) has been used in several European Space Agency (ESA) and Japan Aerospace Exploration Agency (JAXA) GCF investigations on the International Space Station.
The Granada Crystallization Facility (GCF) was designed to optimize the number of experiments, reduce the volume of protein solutions, and reduce the cost of the crystallization in space. The GCF holds the Granada Crystallization Box (GCB), which consists of three parts—box body, capillary holder, and cover—that are made of polystyrene. The box body is a narrow box that is open on one side. The box is small enough to stow and transport the experiments, yet able to hold as many as six different crystallization experiments. It is transparent and narrow to allow microscopic observation of the experiments. The capillary holder fits into the box body; the design rationale was to maximize the density of experiments without compromising either easy insertion and handling of the capillaries or microscopic observation capabilities. The capillary holder can accommodate capillaries ranging from 0.1 to 1.5 mm in diameter. The holder was designed as a separate part to increase flexibility; the design allows procedures other than counterdiffusion to be performed within the box body. The parts were constructed of polystyrene for optical quality, chemical stability, physical stability, and price.
Counterdiffusion crystallization experiments have been performed in the GCB. The bottom of the box contains a layer of buffered agarose gel at a 0.5 percent w/v concentration. Capillaries with diameters ranging from 0.2 to 1.5 mm are used as protein chambers. Once the gel is set, the capillaries are filled with the protein, inserted through the hole of the capillary holder, and inserted at a given depth into the gelled buffer layer. This depth represents the effective width of the buffer layer for passive activation and will be calculated as a function of the time elapsed between the time the reactor is filled and the time it is placed in its final location in the International Space Station (ISS). Finally the salt solution is poured on top of the buffer gel.
The GCB can be used as a passive reactor without moving parts or crew manipulation for microgravity experiments. The method is simple. The crystallization reactors must be supplied for integration into the spacecraft several hours before launch (the waiting time for launch). After takeoff, it takes time to reach orbit around the Earth (about 8.5 minutes) and, in the case of the ISS, to be locked to the ISS and place the crystallization reactor in the proper location (the waiting time for orbit). Taking into account these considerations, the capillaries are punching into the gel with a depth x such that x > (Dt)1/2, where "D" is the diffusion coefficient of the precipitating agent and "t" is the waiting time for launch plus the waiting time for orbit. As a result, during the waiting times for launch and orbit, the precipitating agent is diffusing across the gel, and convection is precluded. Once the GCB is orbiting the Earth, the precipitating agent will reach the protein solution filling the capillaries, and crystallization will take place in the gel-free solution under diffusion-controlled mass transport. Therefore, the experiment in the GCF can be considered totally passive, with no activation and/or manipulation by crewmembers required.
Protein crystallization experiments in the International Space Station were performed using Granada Crystallization Boxes (GCBs), each of which accommodates up to six capillary counterdiffusion experiments.
The Granada Crystallization Box (GCB) is used to perform counterdiffusion experiments. The GCB consists of three elements made of polystyrene: a box into which the gel is introduced, a capillary holder to hold capillaries, and a cover. The buffer layer is applied at the bottom of the reservoir after the capillary holder is inserted. Once the gel is set, the capillaries are filled with the protein, inserted through the hole of the capillary holder, and inserted to a given depth into the gelled buffer layer. The salt solution is set on top of the buffer gel.
The GCF is a passive device. The hardware requires no crew time on orbit except the time it takes to place and remove the GCF.
Protein crystallization experiments on the International Space Station (ISS)were performed on the Andromede and Odissea missions using 23 Grenada Crystallization Boxes (GCBs). Each GCB accommodates as many as six capillary counterdiffusion experiments.
The Grenada Crystallization Facility (GCF) is an aluminium container with internal dimensions of 12 x 12 x 8 cm. The GCFs for the GCBs were designed, built, and tested to ensure that they were capable of withstanding launch and reentry maneuvers and met ISS safety requirements.
The preliminary analysis of the data sets collected with synchrotron radiation shows that the crystals grown using the counterdiffusion technique share excellent global indicators of x-ray data quality, with no obvious difference between crystals grown in reduced-convection conditions in space and crystals grown in convection-free conditions on the ground.
Wyns L, Van de Weerdt C, Martial J, Vanhee C, Evrard C, Maes D, Zegers I, Declercq J. TIM Crystals Grown by Capillary Counterdiffusion: Statistical Evidence of Quality Improvement in Microgravity. Crystal Growth & Design. 2007; 7(11): 2161-2166. DOI: 10.1021/cg700687t.
Tanaka H, Inaka K, Sugiyama S, Sato M, Takahashi S, Yoshitomi S, Sano S. A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions. Journal of Synchrotron Radiation. 2004; 11: 45-48.
Garcia Ruiz JA, Gavira-Gallardo JAntonio, Gonzalez Ramirez LAntonio, Otalora Munoz F. Granada Crystallisation Box: a new device for protein crystallization by counter-diffusion techniques. Acta Crystallographica. 2002; Section D58: 1638-1642.
Garcia-Ruiz JManuel. Nucleation of protein crystals. Journal of Structural Biology. 2003; 142: 22-31.
Ng JD, Gavira-Gallardo JAntonio, Garcia-Ruiz JManuel. Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. Journal of Structural Biology. 2003; 142: 218-231.
Evrard C, Carotenuto L, Dubois F, Zegers I, Garcia-Ruiz JManuel, Legros J, Otalora Munoz F, De Gieter P, Gonzales-Ramires L, Istasse E, Martial J, Minetti C, Queeckers P, Cedric S, Van de Weerdt C, Willaert R, Wyns L, Yourassowsky C. Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (Protein Microscope for the International Space Station). Microgravity Science and Technology. 2006; 18-3/4: 165-169.