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Facility/Payload OverviewBrief Facility Summary
The Granada Crystallization Facility (GCF) is a multi-user facility designed to conduct crystallization experiments of biological macromolecules in microgravity using a counter-diffusion technique inside capillaries. The counterdiffusion technique is implemented in the Granada Crystallization Boxes (GCB).
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|17|
Previous ISS MissionsThe GCF has been used on several ISS Expeditions for the ESA-GCF and JAXA-GCF investigations.
Facility/Payload Description
Facility Summary
- The Granada Crystallization Facility (GCF) has been designed to crystallize macromolecules in space at a very low cost assuring maximum success.
- The GCF is autonomous, it does not require crew time to be operated and it is a passive device (it does not need electric power to work). GCF can hold 300 crystallization experiments with a volume of one-liter and a mass of approximately 1-kg.
- GCF works by counterdiffusion technique, to minimize the effect of (surface driven) convection and g-jitter on the experiments. In addition to their low cost the GCF has the following advantages:
- it works under diffusion-controlled mass transport
- the counterdiffusion technique automatically searches for the optimal crystallization conditions
- volume of protein required for the experiment is minimized.
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 space crystallization. The GCF holds the Granada Crystallization Box (GCB); the GCB consists of three parts made of polystyrene: the box body consisting of a narrow box open on one side, the capillary holder that fits into the body and the cover lid that closes the box. The box body has been designed to be small enough for experiment stowage and transport yet being able to hold up to six different crystallization experiments. The box is transparent and narrow to allow microscopic observation of the experiments. The rationale behind the design of the capillary holder is as follows: the density of experiments must be maximized but without compromising neither easy insertion and handling of capillaries nor the microscopic observation capabilities. The capillary holder can accommodate capillaries of diameter ranging from 0.1 mm to 1.5 mm. The construction of the handler as a separate part was decided for flexibility because this design allows the implementation of techniques other than counterdiffusion in capillaries within the same box body. Polystyrene was used as the building material balancing the benefits and drawbacks of different materials for optical quality, chemical stability, physical stability and price.
Counter-diffusion crystallization experiments are performed in the GCB. The bottom of the body of the box was filled with a layer of buffered agarose gel at 0.5 percent w/v concentration. Capillaries of different inner diameter ranging from 0.2 to 1.5 mm were used as protein chamber. Once the gel is set, the capillaries are filled with the protein and inserted trough the hole of the guide and punctuated to a given depth into the gelled buffer layer. This punctuation depth represents the effective width of the buffer layer for passive activation and will be calculated as a function of the time elapsed between reactor filling and the final location into the ISS. Finally the salt solution is poured on top of the buffer gel.
GCB can be used for microgravity experiments as a passive reactor, without any moving part or crew manipulation. The method is simple. To perform microgravity experiments the crystallisation reactors must be supplied for integration in the spacecraft a few hours before the launch time (the waiting time for launch). After take-off, there is a time required to reach the orbit around the Earth (about 8 minutes and a half) and (for the case of the International Space Station) to be locked to the ISS and to locate the crystallisation reactor in the proper place (time the waiting time for orbiting). 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" the waiting time for launching plus the waiting time for orbiting. As a result, during the waiting times for launch and orbiting, 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 the crystallization will take place in gel-free solution under diffusion controlled mass transport. Therefore, the experiment in the GCF can be considered as totally passive, no activation and/or manipulation by crewmembers being required.
Operations
Facility Operations
Protein crystallization experiments in the International Space Station were performed using Granada Crystallization Boxes (GCB) each of which accommodate up to six capillary counter-diffusion experiments.
The Granada Crystallization Box (GCB) is a device to perform counter-diffusion experiments. The GCB consists of three elements made of polystyrene; a reservoir to introduce the gel, a guide to hold capillaries, and a cover. The buffer layer is located at the bottom of the reservoir after inserting the guide. Once the gel is set, the capillaries are filled with the protein and inserted through the hole of the guide and punctuated to given depth into the gelled buffer layer. Finally the salt solution is set on top of the buffer gel.
GCF is a passive device. No crew time was required on orbit, except the location and removal of the GCF.
Results/More Information
Protein crystallization experiments in the ISS were performed using twenty-three GCBs on the Andromede and Odissea missions each of which accommodate up to six capillary counter-diffusion experiments. The GCF for the GCBs were designed, built, and tested, capable of withstanding the launch and re-entry maneuver, and that meets the safety requirements of the ISS. The GCF, consist of an aluminium container which has internal dimensions of 12 x 12 x 8 cm. Regarding the crystal quality, the preliminary analysis of the data sets collected with synchrotron radiation shows that the crystals grown with the counter-diffusion technique share excellent global indicators of x-ray data quality with no obvious difference between crystals grown under reduced convection conditions in space and crystals grown under convection free conditions on ground.
Availability
Related Web Sites
Publications
Results Publications
Related Publications
- Zegers I, Carotenuto L, Evrard C, Garcia-Ruiz JM, De Giter P, Gonzalez-Ramirez L, Istasse E, Legros JC, Martial J, Minetti C, Otalora F, Queeckers P, Schockaert C, VandeWeerdt C, Willaert R, Wyns L, Yourassowsky C, Dubois F. Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (Protein Microscope for the International Space Station). Microgravity, Science and Technology, Z-Tec Publishing. ;XVIII-3/4: 165-169. 2006
- Tanaka H, Inaka k, Sugiyama S, Takahashi S, Sano S, Sato M, Yoshitomi S. A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions. Journal of Synchrotron Radiation. ;11:45-48. 2004
- Garcia-Ruiz JM. Nucleation of protein crystals. Journal of Structural Biology. ;142: 22-31. 2003
- Ng JD, Gavira JA, Garcia-Ruiz JM. Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. Journal of Structural Biology. ;142: 218-231. 2003
- Garcia Ruiz JA, Gonzalez-Ram?rez LA, Gavira JA, Otalora F. Granada Crystallisation Box: a new device for protein crystallization by counter-diffusion techniques. Acta Crystallographica. ;Section D58: 1638-1642. 2002
- Evrard C, Maes D, Zegers I, Declercq JP, Vanhee C, Martial J, Wyns L, Van De Weerdt C. TIM Crystals Grown by Capillary Counterdiffusion: Statistical Evidence of Quality Improvement in Microgravity. Crystal Growth and Design. ;7: 2161-2166. 2007
- Maes D, Gonzalez-Ramirez LA, Lopez-Jaramillo J, Yu B, De Bondt H, Zegers I, Afonina E , Garcia-Ruiz JM, Gulnik S. Structural study of the type II 3-dehydroquinate dehydratase from Actinobacillus pleuropneumoniae. Acta Crystallographica. ;Section D60: 463-471. 2004
Images
Granada crystallization box hardware, image courtesy of Triana Science and Technology, Granada, Spain.+ View Larger Image
NASA Image: ISS003E5331 - Expedition Three Flight Engineer Vladimir N. Dezhurov unpacks a bag with the Granada Crystallization Facility the Zvezda, Service Module during the Andromede mission.+ View Larger Image
NASA Image: ISS003E5394 - The Expedition 3 crewmembers, Flight Engineer Mikhail Tyurin (left), Mission Commander Frank L. Culbertson (center) and Flight Engineer Vladimir Dezhurov (right), assemble for a group photo in the Zvezda, Service Module of the ISS. In the background the Granada Crystallization Facility is visible in the orange container.+ View Larger Image
Information Provided and Updated by the ISS Program Scientist's Office