how gcb works in space crystallisation
DESCRIPTION
How GCB works in space crystallisation. Juan Ma. Garcia-Ruiz Laboratorio de Estudios Cristalográficos. The Granada Crystallisation Box consists of three elements:. A reservoir to introduce the gel. capillary. A guide holding the capillaries. A cover. gel. 0.1 %. Experimental design. - PowerPoint PPT PresentationTRANSCRIPT
How GCB works in space crystallisation
Juan Ma. Garcia-RuizLaboratorio de Estudios Cristalográficos
The Granada Crystallisation Box consists of three elements:
A reservoir to introduce the gel
capillary
gel
A guide holding the capillaries
A cover
Use of GCB in space Experimental design
[Protein] = 0
[Precipitant] = nP
[Adittives] = A
Implementation on-ground
Implementation in space
[Protein] = 0
[Precipitant] = P
[Adittives] = A
[Protein] = C
[Precipitant] = P
[Adittives] = A
Capillary diameter : from 0.2 mm to 1.0 mm
0.1 %
0 %
0 %
1 % 1 %
0.1 %
0 %
In yellow
% of agarose
t = -8 h
How GCB works in Space
During the waiting time for launch, the precipitating agent diffuse across the gel layer
t = 0 h
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.
How GCB works in Space
t 48 h
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.
How GCB works in Space
2d < t < 40d
During the stage at the International Space Station, the proteins crystals form inside the capillaries.
How GCB works in Space
t = 40 d
The GCF returns to the Earth.
How GCB works in Space
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
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
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.
HEW Lysozyme
= 0.3 mm
Dehydroquinase
= 1.0 mm
Concanavalin A
= 0.4 mm
Thaumatin
= 1.0 mm
CabLys3*lysozyme
= 0.5 mm
Catalase
= 0.2 mm
Some crystals grown during the GCF test in the Andromede mission
Use of the GCB in space
Use of GCB in space Results
X-ray DiffractionDehydroquinase
0
10
20
30
40
50
60
70
80
90
1,6 1,8 2 2,2 2,4 2,6 2,8 3 3,2 3,4 3,6 3,8 4Resolution (A)
I/s
Space Ground structural Dataset (ground)
Catalase
01020304050607080
1,4 1,6 1,8 2 2,2 2,4 2,6 2,8 3 3,2 3,4 3,6 3,8 4
Resolution (A)
I/s(I)
Ground Space Structural Dataset
Dehydroquinase Best crystals by other techniques: 3.5 Å
Space Ground Structural purposes (Ground)
Beam Line EMBL-Hamburg
BW7B BW7B BW7B
Wave length (Å) 0.8463 0.8463 0.8463 Distance to detector (mm) 270 270 270 Oscillation angle 0.3 0.3 0.3 Data collection Temperature
100 K 100 K 100 K
Space group P222 P222 P222
Unit cell a b c == = 90
129.09 131.33 161.62
128.72 131.25 160.72
128.96 131.35 160.84
Mosaicity by XDS 0.1 0.11 0.11 Resolution range (Å) 10.00 – 1.71 10.00 – 1.71 10.00 – 1.71 Completeness 71.5 59.5 98.2 Multiplicity 1.6 2.0 5.1 Rsym 3.7 2.2 3.5 I/(I) 12.7 20.3 27.8 Outer resolution shell (Å) 1.80 – 1.71 1.80 – 1.71 1.80 – 1.71 Completeness 66.2 59.4 96.0
Catalase Best crystals by other techniques: 3.4 Å
Space Ground Structural purposes
Beam Line EMBL-Hamburg
X13 X13 X13
Wave length (Å) 0.801 0.801 0.801 Distance to detector (mm) 150 / 240 150 / 240 150 / 240 Oscillation angle 0.6 0.6 0.6 Data collection Temperature
100 K 100 K 100 K
Space group
P3121 P3121 P3121
Unit cell a=b c == 90 = 120
142.2 175.0
142.3 175.1
142.26 175.03
Mosaicity by XDS 0.145 0.140 Resolution range (Å) 15.0 – 1.6 15.0– 1.6 15.0 – 1.6 Completeness 88.2 80.2 91.5 Multiplicity 2.4 2.7 4.7 Rmerge 2.7 3.1 3.7 I/(I) 17.7 15.6 21.5 Outer resolution shell (Å) 1.8 – 1.6 1.8 – 1.6 1.8 – 1.6 Completeness 72.2 66.9 78.5
Use of GCB in space Results
X-ray Diffraction
Lysozyme Best crystals by other Techniques: 0.97 Å
Space Ground
Beam Line EMBL-Hamburg
BW7B BW7B
Wave length (Å) 0.8463 0.8463 Distance to detector (mm) 130 130 Oscillation angle 1 / 1.4 1 / 1.3 Data collection Temperature
100 K 100 K
Space group P43212 P43212
Unit cell a=b c ==
78.73 36.95 90.00
78.72 36.97 90.00
Mosaicity by XDS 0.09 0.09 Resolution range (Å) 1.40 – 0.95 1.40 – 0.95 Completeness 68.3 66.6 Multiplicity 2.7 2.3 Rsym 5.4 6 I/(I) 19.2 20.2 Outer resolution shell (Å) 1.00 – 0.94 1.00 – 0.94 Completeness 76.9 70.1 Rsym 19.2 20.2
Lys_high
05
1015202530
0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45
Resolution (A)
I/s(I)
Space ground
Use of GCB in space Results
X-ray Diffraction
Thaumatin Space+Gel Space Ground+Gel Space+Gel Space Beam Line EMBL-Hamburg
BW7B BW7B BW7B BW7B BW7B
Wave length (Å) 0.8463 0.8463 0.8463 0.8463 0.8463 Detector distance (mm) 200 200 200 200 200 Oscillation angle 0.5 0.5 0.5 1 1 Data collection Temperature
Room temperature
Room temperature
Room temperature
100 K 100 K
Space group
P41212 P41212 P41212 P41212 P41212
Unit cell a=b c ==
58.612 151.690
90
58.655 151.644
90
57.651 151.637
90
57.683 149.902
90
57.693 149.963
90 Mosaicity by XDS 0.09 0.095 0.095 0.1 0.1 Resolution range (Å) 15 - 1 15 –1 15 – 1 10 –1 10 –1 Completeness 69-33 64.1 68.6 84.1 84 Multiplicity 2.0 2.1 2.0 2.6 2.4 Rsym 3.0 3.4 2.6 5.1 3.2 I/(I) 10.6 9.3 12.3 12.0 17.5 Outer resolution shell (Å) 1.1 – 1.0 1.1 – 1.0 1.1 – 1.0 1.1 – 1.0 1.1 – 1.0 Completeness 39.9 34.7 39.6 57.2 58.9
Thaumatin room T
0
10
20
30
40
50
60
1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
Resolution (A)
I/sigma(I)
Space+Gel Space Ground+Gel
Thaumatin 100 K
0
10
20
30
40
50
1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8
Resolution (A)
I/sigma(I)
Space+Gel Space
Use of GCB in space
Conclusions1. The results validate the GCB for space experiments as a passive,
inexpensive and high-density crystallisation facility for growing protein crystals.
2. 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.
3. 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.
A cooperation philosophy:
LEC (Granada) team, with NTE and Mars Center, supply:
The facility (GCF) to be used in spaceThe reactors (GCB) to perform the experimentsThe gel to be used in the experimentsThe preparation of the experiments at the launch siteThe help for properly preparing counter-diffusion experiments
The participanting laboratories contribute by:
supplying the proteinsPerforming preliminary experiments to tune the crystallisation conditionsEvaluation the crystal quality of on-ground- and space grown crystals
The obtained crystals and diffraction data remain the property of the participating laboratories.
Use of GCB in space Andromede
mission1. Alliinase (Institute for Molecular Biotechnology, Jena, Germany)
2. CabLys3*lysozyme (Institute of Mol. Biol. Biotechn., Brussels, Belgium)
3. Caf1M (Institute of Inmunological Engineering, Chekhov District, Russia)
4. Catalase (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia)
5. Concanavalin A (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain)
6. Cytochrome C (Institute of Chemical and Biological Tecnology, Oeiras, Portugal)
7. Dehydroquinase (DHQ) (Tibotec-Virco, Mechelen, Belgium)
8. Endo VII (European Molecular Biology Laboratory (EMBL), Heidelberg, Germany)
9. Factor XIII (Institute for Molecular Biotechnology, Jena, Germany)
10. Ferritin (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain)
11. Gamma-E-crystallin (European Molecular Biology Lab. (EMBL), Grenoble, France)
12. HEW Lysozyme (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain)
13. Leghemoglobin (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia)
14. Low density Lipoprotein (LDL) (University Hospital of Freiburg, Freiburg, Germany)
15. Lumazine synthase (Technische Universitaet Muenchen, Garching, Munich, Germany)
16. Propeptide of Cathepsin S (Institute for Molecular Biotechnology, Jena, Germany)
17. RNAse II (Institute of Chemical and Biological Technology, Oeiras, Portugal)
18. Saicar-synthase (A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russia)
19. Sm-like protein (European Molecular Biology Lab. (EMBL), Heidelberg, Germany)
20. S-COMT (Institute of Chemical and Biological Technology, Oeiras, Portugal)
21. Thermus thermophilus EF-Tu (Institute for Molecular Biotechnology, Jena, Germany)
22. Thaumatin (Laboratorio de Estudios Cristalográficos (LEC), Granada, Spain)
GCB PROTEIN LABORATORY
GCB-01
Pike Parvalbumin Prof. J. P. Declercq, University of Louvain, Louvain-la-Neuve, BELGIUMGCB-02
GCB-03
GCB-04
Triosephosphate isomerase Prof. Martial, Universite de Liege, Liege, BELGIUMGCB-05
GCB-06
GCB-07
(Pro-Pro-Gly)10 Prof. A. Zagari, University of Naples, Napoli, ITALYGCB-08
GCB-09
GCB-10
Camel VHH antibody fragment Prof. L. Wyns, Vrije Universiteit Brussel, Brussels, BELGIUMGCB-11
GCB-12
GCB-13 -AmylaseProf. H. Komatsu, NASDA, Ibaraki, JAPAN
GCB-14 Lysozyme
GCB-15 Bacterial antiinfectivity protein Allan D’Arcy, Morphochem, Schwarzwaldallee, SWITZERLAND
GCB-16 AF-Sm1complexed with RNA
Prof. D. Suck, EMBL, Heidelberg, GERMANYGCB-17 Endonuclease VII from Phage T4
GCB-18 Hfq from E. Coli
GCB-19 Gamma-CProf. D. Myles, EMBL, Grenoble, FRANCE
GCB-20 Gamma-E
GCB-21 Low Density Lipoprotein Prof. M. Baumstark, Medizinische Univ. Freiburg, Freiburg, GERMANY
GCB-22 TrypsinProf. J.M. Garcia-Ruiz, LEC, CSIC-UGR, Granada, SPAIN
GCB-23 Lysozyme