How GCB works in space crystallisation

1. How GCB works in space crystallisation Juan Ma. Garcia-Ruiz Laboratorio de Estudios Cristalográficos

2. The Granada Crystallisation Box consists of three elements: A reservoir to introduce the gel capillary A guide holding the capillaries A cover gel

3. 0.1 % Experimental design Use of GCB in space Implementation on-ground Implementation in space [Protein] = C [Precipitant] = P [Adittives] = A 0 % [Protein] = 0 [Precipitant] = nP [Adittives] = A 0.1 % 0 % 0 % [Protein] = 0 [Precipitant] = P [Adittives] = A 1 % 1 % Capillary diameter : from 0.2 mm to 1.0 mm In yellow % of agarose

4. How GCB works in Space During the waiting time for launch, the precipitating agent diffuse across the gel layer t = -8 h

5. How GCB works in Space Vibrations during the launch are buffered by the gel where the capillaries are punched. The capillaries are oriented perpendicular to g. The precipitating agent continue to diffuse across the gel. t = 0 h

6. How GCB works in Space The penetration length of the capillaries can be calculated so that the protein starts to cristalise into the capillaries once in the ISS After eight minutes and a half, the vehicle is under free fall. t  48 h

7. How GCB works in Space During the stage at the International Space Station, the proteins crystals form inside the capillaries. 2d < t < 40d

8. How GCB works in Space The GCF returns to the Earth. t = 40 d

9. Simulation Use of GCB in space Fluid Dynamic Computer Simulation Fixed parameters: Capillary diameter = 0.7 mm H gel layer = 2.7 cm Length of the box = 3.3cm H salt layer = 5.3 cm Width of the box = 0.4 cm H punctuation = 1 cm Protein diffusion coefficient = 1.16 x 10-6 cm2/s Salt diffusion coefficient = 2.338 x 10-19 cm2/s Ratio Ksp/Ks = 3 Variables: [Lisozyme]i = 100 – 50 – 30 mg/mL [NaCl]i = 20 – 10- 15 % Protein height in the capillary = 4 – 5 – 6 cm Front of Growth

10. Results Use of GCB in space GCB Validation as a Flight Facility • None of the GCBs suffered any damage • All the capillaries remained in position • None of the gels were broken • No leakage occured that could affect the physicochemical conditions of the experiment • Whenthere were no crystals from space there werenone in theon-groundexperiment, either, and vice versa

11. Use of the GCB in space The dimensions of the GCF (13 cm x 13 cm x 8 cm), its weight on ground (1 kilogram), and the number of capillary experiments it can accommodate (138) make the GCF be the cheapest, simple and efficient instrument for applied protein crystallisation in space. Some crystals grown during the GCF test in the Andromede mission = 1.0 mm = 0.4 mm = 0.2 mm Catalase Dehydroquinase Concanavalin A = 1.0 mm = 0.5 mm = 0.3 mm Thaumatin HEW Lysozyme CabLys3*lysozyme

12. Results Use of GCB in space X-ray Diffraction

13. Results Use of GCB in space X-ray Diffraction

14. Results Use of GCB in space X-ray Diffraction

15. Use of GCB in space Conclusions • The results validate the GCB for space experiments as a passive, inexpensive and high-density crystallisation facility for growing protein crystals. • From the point of view of resolution limit, there are no obvious differences between crystals grown under reduced convective flow in space and crystals grown under convection free conditions on ground. The crystals grown with the counter-diffusion technique share excellent global indicators of X-ray quality. The counter-diffusion technique can be implemented in two ways: One is in space, where the absence of gravity avoids convection and allows the diffusive environment required for our technique. The other way to get the same diffusive environment on ground is the use of gels, but obviously, the gel may interfere with the chemicals used in crystallisation. We are in the evaluation phase of both possible implementations.

16. A cooperation philosophy: • LEC (Granada) team, with NTE and Mars Center, supply: • The facility (GCF) to be used in space • The reactors (GCB) to perform the experiments • The gel to be used in the experiments • The preparation of the experiments at the launch site • The help for properly preparing counter-diffusion experiments • The participanting laboratories contribute by: • supplying the proteins • Performing preliminary experiments to tune the crystallisation conditions • Evaluation the crystal quality of on-ground- and space grown crystals • The obtained crystals and diffraction data remain the property of the participating laboratories.

17. Use of GCB in space Andromede mission • Alliinase (Institute for Molecular Biotechnology, Jena, Germany) • CabLys3*lysozyme (Institute of Mol. Biol. Biotechn., Brussels, Belgium) • Caf1M (Institute of Inmunological Engineering, Chekhov District, Russia) • Catalase (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia) • Concanavalin A (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain) • Cytochrome C (Institute of Chemical and Biological Tecnology, Oeiras, Portugal) • Dehydroquinase (DHQ) (Tibotec-Virco, Mechelen, Belgium) • Endo VII (European Molecular Biology Laboratory (EMBL), Heidelberg, Germany) • Factor XIII (Institute for Molecular Biotechnology, Jena, Germany) • Ferritin (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain) • Gamma-E-crystallin (European Molecular Biology Lab. (EMBL), Grenoble, France) • HEW Lysozyme (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain) • Leghemoglobin (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia) • Low density Lipoprotein (LDL) (University Hospital of Freiburg, Freiburg, Germany) • Lumazine synthase (Technische Universitaet Muenchen, Garching, Munich, Germany) • Propeptide of Cathepsin S (Institute for Molecular Biotechnology, Jena, Germany) • RNAse II (Institute of Chemical and Biological Technology, Oeiras, Portugal) • Saicar-synthase (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia) • Sm-like protein (European Molecular Biology Lab. (EMBL), Heidelberg, Germany) • S-COMT (Institute of Chemical and Biological Technology, Oeiras, Portugal) • Thermus thermophilus EF-Tu (Institute for Molecular Biotechnology, Jena, Germany) • Thaumatin (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain)