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
Experimental design
Use of GCB in space
Implementation
on-ground
Implementation
in space
[Protein] = C
[Precipitant] = P
[Adittives] = A
0 %
[Protein] = 0
0.1 %
0.1 %
[Precipitant] = nP
0 %
[Adittives] = A
0 %
[Protein] = 0
1 %
In yellow
% of agarose
[Precipitant] = P
[Adittives] = A
Capillary diameter : from 0.2 mm to 1.0 mm
1 %
How GCB works in Space
During the waiting time for launch, the precipitating
agent diffuse across the gel layer
t = -8 h
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=0h
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
How GCB works in Space
During the stage at the
International Space
Station, the proteins
crystals form inside the
capillaries.
2d < t < 40d
How GCB works in Space
The GCF returns to the
Earth.
t = 40 d
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
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
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
= 0.2 mm
Catalase
= 1.0 mm
Thaumatin
= 1.0 mm
Dehydroquinase
= 0.5 mm
CabLys3*lysozyme
= 0.4 mm
Concanavalin A
= 0.3 mm
HEW Lysozyme
Results
Use of GCB in space
X-ray Diffraction
Dehydroquinase
I/s
Catalase
Dehydroquinase
Space
Best
crystals
by other Space
Best
crystals
by other
techniques:
3.5 3.4
Å Å
techniques:
90
80
70
60
50
40
30
20
10
0
1,6 1,8
Space
2
2,2 2,4 2,6 2,8
Ground
3
3,2 3,4 3,6 3,8 4
Resolution (A)
structural Dataset (ground)
Catalase
I/s(I)
80
70
60
50
40
30
20
10
0
1,4
1,6
Ground
1,8
2
2,2
Space
2,4
2,6
2,8
3
3,2
Structural Dataset
3,4
3,6
3,8
4
Resolution (A)
Ground
Ground
Beam
LineLine
Beam
BW7B
BW7B
X13
X13
EMBL-Hamburg
EMBL-Hamburg
Wave
length
(Å) (Å)
0.8463
0.8463
Wave
length
0.801
0.801
Distance
to
detector
(mm)
270
270
Distance to detector (mm)
150 / 240
150 / 240
Oscillation
angle
0.3 0.6
0.30.6
Oscillation
angle
DataData
collection
collection
100 100
K K
100
KK
100
Temperature
Temperature
Space group
Space group
P222
P222
P3121
P3121
Unit cell
a
129.09
128.72
Unit cell b a=b
142.2
142.3
131.33
131.25
175.0
175.1
cc
161.62
160.72
===90
==
90= 120
Mosaicity
by
XDS
0.140
Mosaicity by XDS
0.10.145
0.11
Resolution
range
– 1.610.00
15.0–
1.6
Resolution
range
(Å) (Å)
10.0015.0
– 1.71
– 1.71
Completeness
80.2
Completeness
71.588.2
59.5
Multiplicity
Multiplicity
1.6 2.4
2.02.7
Rmerge
2.7
Rsym
3.7
2.23.1
15.6
12.717.7
20.3
I/(I)
I/(I)
1.71
– 1.71
Outer
resolution
shellshell
(Å) (Å)1.80 –1.8
– 1.6 1.801.8
– 1.6
Outer
resolution
Completeness
66.272.2
59.4
Completeness
66.9
Structural
Structural
purposes
purposes
(Ground)
BW7B
X13
0.8463
0.801
150270
/ 240
0.3
0.6
100 K
K
100
P222
P3121
128.96
142.26
131.35
175.03
160.84
0.11
15.0
10.00 –– 1.6
1.71
91.5
98.2
4.7
5.1
3.7
3.5
21.5
27.8
1.80
1.71
1.8 – 1.6
96.0
78.5
Results
Use of GCB in space
X-ray Diffraction
Lys_high
I/s(I)
Lysozyme
Best crystals by other
Techniques: 0.97 Å
30
25
20
15
10
5
0
0.9 0.95
Space
1
1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45
ground
Resolution (A)
Beam Line
EMBL-Hamburg
Wave length (Å)
Distance to detector (mm)
Oscillation angle
Data collection
Temperature
Space group
Unit cell
a=b
c
==
Mosaicity by XDS
Resolution range (Å)
Completeness
Multiplicity
Rsym
I/(I)
Outer resolution shell (Å)
Completeness
Rsym
Space
Ground
BW7B
BW7B
0.8463
130
1 / 1.4
0.8463
130
1 / 1.3
100 K
100 K
P43212
P43212
78.73
36.95
90.00
0.09
1.40 – 0.95
68.3
2.7
5.4
19.2
1.00 – 0.94
76.9
19.2
78.72
36.97
90.00
0.09
1.40 – 0.95
66.6
2.3
6
20.2
1.00 – 0.94
70.1
20.2
Results
Use of GCB in space
X-ray Diffraction
Thaumatin room T
I/sigma(I)
Thaumatin 100 K
I/sigma(I)
60
50
50
40
40
30
30
20
20
10
10
0
0
1
1.2
Space+Gel
1.4
1.6
1.8
Space
2
2.2
Ground+Gel
2.4
2.8
1
Resolution (A)
Thaumatin
Beam Line
EMBL-Hamburg
Wave length (Å)
Detector distance (mm)
Oscillation angle
Data collection
Temperature
Space group
Unit cell
2.6
a=b
c
==
Mosaicity by XDS
Resolution range (Å)
Completeness
Multiplicity
Rsym
I/(I)
Outer resolution shell (Å)
Completeness
1.2
1.4
Space+Gel
1.6
1.8
2
Space
2.2
2.4
2.6
2.8
Resolution (A)
Space+Gel
Space
Ground+Gel
Space+Gel
Space
BW7B
BW7B
BW7B
BW7B
BW7B
0.8463
200
0.5
Room
temperature
0.8463
200
0.5
Room
temperature
0.8463
200
0.5
Room
temperature
0.8463
200
1
0.8463
200
1
100 K
100 K
P41212
P41212
P41212
P41212
P41212
58.612
151.690
90
0.09
15 - 1
69-33
2.0
3.0
10.6
1.1 – 1.0
39.9
58.655
151.644
90
0.095
15 –1
64.1
2.1
3.4
9.3
1.1 – 1.0
34.7
57.651
151.637
90
0.095
15 – 1
68.6
2.0
2.6
12.3
1.1 – 1.0
39.6
57.683
149.902
90
0.1
10 –1
84.1
2.6
5.1
12.0
1.1 – 1.0
57.2
57.693
149.963
90
0.1
10 –1
84
2.4
3.2
17.5
1.1 – 1.0
58.9
Use of GCB in space
Conclusions
1.
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
global indicators of X-ray quality.
share excellent
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 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.
Use of GCB in space
Andromede mission
1.
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
GCB-02
Pike Parvalbumin
Prof. J. P. Declercq, University of Louvain, Louvain-la-Neuve, BELGIUM
Triosephosphate isomerase
Prof. Martial, Universite de Liege, Liege, BELGIUM
(Pro-Pro-Gly)10
Prof. A. Zagari, University of Naples, Napoli, ITALY
Camel VHH antibody fragment
Prof. L. Wyns, Vrije Universiteit Brussel, Brussels, BELGIUM
GCB-03
GCB-04
GCB-05
GCB-06
GCB-07
GCB-08
GCB-09
GCB-10
GCB-11
GCB-12
GCB-13
-Amylase
GCB-14
Lysozyme
GCB-15
Bacterial antiinfectivity protein
GCB-16
AF-Sm1complexed with RNA
GCB-17
Endonuclease VII from Phage T4
GCB-18
Hfq from E. Coli
GCB-19
Gamma-C
GCB-20
Gamma-E
GCB-21
Low Density Lipoprotein
GCB-22
Trypsin
GCB-23
Lysozyme
Prof. H. Komatsu, NASDA, Ibaraki, JAPAN
Allan D’Arcy, Morphochem, Schwarzwaldallee, SWITZERLAND
Prof. D. Suck, EMBL, Heidelberg, GERMANY
Prof. D. Myles, EMBL, Grenoble, FRANCE
Prof. M. Baumstark, Medizinische Univ. Freiburg, Freiburg, GERMANY
Prof. J.M. Garcia-Ruiz, LEC, CSIC-UGR, Granada, SPAIN