Use of Granada Crystallisation Box GCB

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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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Introduction
Use of GCB in space
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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Objectives
Use of GCB in space

• 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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Objectives
Use of GCB in space
Selected Proteins I

• Alliinase (Institute for Molecular
  Biotechnology, Jena, Germany)
• CabLys3lysozyme (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)

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Objectives
Use of GCB in space
Selected Proteins II

• 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)

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Experiment design
Use of GCB in space
GROUND
SPACE
Time 0
Protein C Precipitating agent
P Additives A
Protein 0 Precipitating agent
nP Additives A
Protein 0 Precipitating agent
P Additives A
Capillary sizes from 0.2 mm up to 1.0 mm
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Experiment design
Use of GCB in space
GROUND
SPACE
Time gt 0
Protein C Precipitating agent
P? Additives A
Protein 0 Precipitating agentnP? Additives
A
Protein 0 Precipitating agent
P? Additives A
Capillary sizes from 0.2 mm up to 1.0 mm
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Grashof number

Use of GCB in space
Values of the Grashof number as a function of the
characteristic length of the system
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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 Lisozymei 100 50 30
mg/mL NaCli 20 10- 15
Protein height in the capillary 4 5 6 cm

Front of Growth
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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 Lisozymei 100 50 30
mg/mL NaCli 20 10- 15
Protein height in the capillary 4 5 6 cm

End of Growth
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Experiment design
Use of GCB in space
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Experiment design
Use of GCB in space
How GCB works in Space
t -8 h
t 0 h
t ? 48 h
2d lt t lt 72d
t 72 d
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Experiment design
Use of GCB in space
How GCB works in Space
Dimensions 13 cm x 13 cm x 8 cm Up to 138
capillaries
t -8 h
t 0 h
t ? 48 h
2d lt t lt 72d
t 72 d
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Experiment design
Use of GCB in space
How GCB works in Space
Time in space 72 days
t - 8 h
t 0 h
t ? 48 h
2d lt t lt 72d
t 72 d
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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
• When there were no crystals from space there
  were none in the on-ground experiment, either,
  and vice versa

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Results
Use of GCB in space
Crystals
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Results
Use of GCB in space
X-ray Diffraction
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Results
Use of GCB in space
X-ray Diffraction
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Results
Use of GCB in space
X-ray Diffraction
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Conclusions
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.

• 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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Final remarks
Use of GCB in space
Thermo-fluid-dynamic regimes from Order of
Magnitude Analysis
Steady Diffusion
Gels (ISS and ground)
Steady Diff.- Conv.
Steady Buoyant boundary layer
ISS ungelled
Unsteady Diffusion
Unsteady Buoyant boundary layer
Unsteady Diff.-conv.
Unsteady Poiseuille layer
?
X-ray diffraction
X-ray diffraction
Would crystals grown from ungelled solutions in
the steady diffusion regime yield better X-ray
diffraction data sets?
How the unsteady diffusion-convection regime
affects the pattern formation in
counter-diffusion experiments?
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?