GCB in Space 4

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Use of Granada Crystallisation Box (GCB) in Space Eva Mañas1, Luis González1, Javier López-Jaramillo1, Dario Castagnolo2, Luigi Carotenuto2 and J.M. Garcia-Ruiz1 1 Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain. 2 Microgravity Advanced Research and Support Center (MARS Centre), Napoli. Italy.

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Use of GCB in space Introduction Franco-Russian Mission Andromede ESTEC Contract Nr. 15338/01/NL/VK Concept and Scientific design: LEC, Granada, Spain Flight Facility : NTE, Barcelona, Spain Computer simulation and MARS Center, Napoli, Italy fluid dynamics analysis: LEC, Granada, Spain

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Use of GCB in space Objectives The tasks performed in the framework of this project were: The design and construction of a passive apparatus (Granada Crystallisation Facility, GCF) to perform counter-diffusion experiments in the ISS inside capillaries using the Granada Crystallisation Box (GCB). To perform preliminary experiments to tune the optimal crystallisation conditions for a wide set of proteins. To analyse the results of the experiments related to the crystallisation environment and kinetics, and to compare them with those of numerical simulations of fluid dynamics. To collect the X-ray diffraction data and to compare the results with those of identical experiments carried out on Earth [To be carried out by the “owner” of the macromolecule].

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Use of GCB in space Selected Proteins I 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) Objectives

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Use of GCB in space 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) Selected Proteins II Objectives

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Use of GCB in space Experiment design [Protein] = 0 [Precipitating agent] = nP [Additives] = A GROUND SPACE [Protein] = 0 [Precipitating agent] = P [Additives] = A [Protein] = C [Precipitating agent] = P [Additives] = A Capillary sizes : from 0.2 mm up to 1.0 mm Time = 0

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Use of GCB in space Experiment design GROUND SPACE [Protein] = 0 [Precipitating agent]=nP [Additives] = A [Protein] = 0 [Precipitating agent] = P [Additives] = A [Protein] = C [Precipitating agent] = P [Additives] = A Capillary sizes : from 0.2 mm up to 1.0 mm Time > 0

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Use of GCB in space Grashof number Values of the Grashof number as a function of the characteristic length of the system

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Use of GCB in space 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 Fluid Dynamic Computer Simulation

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Use of GCB in space Simulation 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 End of Growth

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Use of GCB in space Experiment design

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Use of GCB in space Experiment design t = -8 h t = 0 h t  48 h 2d < t < 72d t = 72 d How GCB works in Space

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Use of GCB in space Experiment design t = -8 h t = 0 h t  48 h 2d < t < 72d t = 72 d Dimensions = 13 cm x 13 cm x 8 cm Up to 138 capillaries How GCB works in Space

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Use of GCB in space Experiment design t = - 8 h t = 0 h t  48 h 2d < t < 72d t = 72 d Time in space = 72 days How GCB works in Space

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Use of GCB in space Results 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 When there were no crystals from space there were none in the on-ground experiment, either, and vice versa

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Use of GCB in space Results Crystals

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Use of GCB in space Results X-ray Diffraction

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Use of GCB in space Results X-ray Diffraction

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Use of GCB in space Results X-ray Diffraction

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Use of GCB in space Conclusions Conclusions The results validate the GCB for space experiments as a passive, inexpensive and high-density crystallisation facility for growing protein crystals. The crystals grown with the counter-diffusion technique share excellent global indicators of X-ray quality. From the point of view of structural resolution, there are no obvious differences between crystals grown under reduced convective flow in space and crystals grown under convection free conditions on ground.

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Use of GCB in space Final remarks Steady Diffusion Unsteady Buoyant boundary layer Steady Diff.- Conv. ISS ungelled Steady Buoyant boundary layer Unsteady Diffusion Unsteady Poiseuille layer Gels (ISS and ground) X-ray diffraction X-ray diffraction  How the unsteady diffusion-convection regime affects the pattern formation in counter-diffusion experiments? Would crystals grown from ungelled solutions in the steady diffusion regime yield better X-ray diffraction data sets? How do gels affect the quality of the crystals depending on concentration and type of gels? Which is the best X-ray data acquisition protocol for comparative studies? Unsteady Diff.-conv. Thermo-fluid-dynamic regimes from Order of Magnitude Analysis