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