The Figure 1: Crystals obtained during USML-2 flight
In the fall of 1995 the APCF(*) flew, as the only European payload, onboard the United States Microgravity Laboratory-2 (USML-2) on Space Shuttle Columbia, space mission STS-73.
(*) A description of the APCF will be found in the following article by W. Riesselmann in this issue of Microgravity News
Lift-off occurred on 20 October, roughly one month after the originally scheduled launch date of 21 September, after six unsuccessful launch attempts, all due to either bad weather or technical difficulties. The Shuttle touched down on the KSC landing strip on 5 November, after almost 16 days in orbit.
It was the third space flight of APCF after Spacehab 1 / STS-57 in 1993 and IML-2 / STS-65 in 1994. Two APCF units were flown on this mission, carrying together a total of 96 experiments in as many protein crystallisation reactors. 12 of these experiments were performed by a group of US investigators from the University of California, Riverside, whilst the remaining 84 experiments had been provided by 14 European groups from seven countries: Belgium, France, Germany, Italy, The Netherlands, Sweden and the United Kingdom.
The purpose of this mission, as of all previous missions of APCF, was to grow bigger and more perfect crystals than could normally be obtained in the laboratory under terrestrial conditions. Investigators would harvest' these crystals after the APCF flight reactors had been returned to them, and they would install the best ones in a special X-ray beam at one of the large European synchroton facilities like DESY (Deutsches Elektronen SYnchroton) in Hamburg (D) or ESRF (European Synchroton Radiation Facility) in Grenoble (F). The radiation patterns obtained from the diffraction of the X-ray beam by the crystals, form the basis of a lengthy and complex process to determine the three- dimensional molecular structure of the respective protein. While in orbit, video images were taken of selected experiments. These images show when and how crystals grow in a microgravity environment.
Investigators are by now busy analysing their mission data. They have compiled and submitted their first preliminary reports. Extracts of these reports will be found in the following.
Principal Investigator: Dr Naomi Chayen, Imperial College, London, England
The protein Apocrustacyanin C is a member of the lipocalin family of proteins, which binds to certain pigments that are widely distributed in plants and animals. Knowledge of the structure of the lipocalins will enable scientists to engineer these proteins to produce carriers that will bind more strongly to the pigment crocetin, which has anticancer properties.
Results:
There is a definite difference between the ground and
flight crystals both in resolution enhancement and in the
internal structures evaluated from rocking curve widths, which
are a measure of the mosaicity of the crystal.
Preliminary Conclusions
There is an indication of improvement in
the space-grown crystals. The amount of improvement is now being
analysed and will be compared with results obtained with lysozyme
experiments performed on the Second International Microgravity
Laboratory mission.
Principal Investigator: Dr Jean-Paul Declercq, Université Catholique de Louvain, Belgium
The bacteriophage Lambda lysozyme is a small protein of 158 amino acids involved in the dissolution of the cell walls of bacteria. Investigators are seeking information about the method of destruction employed by this organism.
Results:
Crystallisation of the protein occurred in two of the
allocated reactors. Several very thin needles were picked out but
appeared to be smaller than the crystals grown on the ground and
were, therefore, unusable for high-resolution X-ray structure
determination and analysis. Precipitates were found in the other
reactors.
Preliminary Conclusions
It was concluded that the crystallisation
conditions of the bacteriophage lambda lysozyme seem to have
changed during the microgravity experiment on USML-2. It can be
assumed that it will be possible to optimise the microgravity
crystallisation parameters to obtain crystals of suitable size
for X-ray analysis.
The Figure 1: Crystals obtained during USML-2 flight
Principal
Investigator: Dr Willem de Grip, University of Nijmegen, The
Netherlands
Visual pigments like rhodopsin are the primary photoreceptor
proteins for a variety of light-regulated processes, such as
vision, circadian entrainment, and photoperiodic reproductivity.
Analysis of the protein crystals is needed if scientists are to
unravel the molecular mechanisms responsible for these processes.
Results:
Some hardware anomalies occurred, and two of the six
reactors partially or completely lost their hanging drops. Two
other reactors did not achieve proper equilibrium, with almost
no reduction in the volume of the protein solution occurring. Of
the remaining two reactors, one had some small crystalline
objects, and the other showed an abundance of small needle-shaped
crystals, including some clusters. All objects were too small
(<80 µm in the largest dimension) to undertake serious diffraction
analysis. Electroimmunoblotting analysis revealed that the
needle-shaped objects contain rhodopsin, but this could not be
unequivocally demonstrated for the other crystal shapes, possibly
because the amount of protein was well below the detection level.
Preliminary Conclusions
These results are less positive than
those obtained with earlier flights. So far, the hardware
problems are unexplained but do not appear to depend on the type
of detergent used. Although crystal sizes are usually smaller in
the ground controls, laboratory-grown crystals tend to grow
larger upon prolonged incubation (4-12 weeks). The results
recommend performance of crystallisation experiments with
rhodopsin under microgravity for longer periods of time (1-3
months).
Principal Investigator: Dr Arnaud Ducruix, CNRS, Université Paris Sud, France
Grb2 is an adaptor protein involved in the transfer of signals from one cell to another. Crystallographers have cloned and analysed Grb2 in ground-based laboratories, but these ground- based crystals do not diffract better than 3.5Å. Better resolution is expected from space-grown crystals. The tetragonal form of lysozyme crystals grown in space on earlier missions was not significantly different from that grown on Earth; however, experiments performed on lysozyme crystals grown in the APCF during the Second International Microgravity Laboratory (IML-2) mission in July 1994 indicated that several crystals were essentially perfect single domains, with rocking curve widths as small as 12 arc seconds (about 3 X 10(exp -3) degrees). (The smaller the value of the rocking curve width, the greater the degree of perfection in the crystal.) Investigators want to extend the study to the triclinic form of lysozyme, expecting to reach values as low as 10(exp -4) degrees.
Results
Analysis in the laboratory showed that the reactors did
not leak, as the salt concentrations were nominal. In addition,
temperature data for the reactors were nominal. All five reactors
maintained near-nominal pH levels, and crystals grew in all
reactors (both flight and ground controls). The APCF reactor
operations, including activation, deactivation, equilibration of
protein/reservoir solution, and thermal regulation, proceeded
successfully. At the time of writing, recorded video images had
not been received yet for analysis. Crystals of both monoclinic
and triclinic lysozyme were immediately mounted in glass
capillaries and were to have been subjected to X-ray radiation
for diffraction limits and mosaicity measurements. Unfortunately,
a long strike in France resulted in the shut-down of the French
Synchrotron Radiation Facility, and the team's allocated beam
time was cancelled.
Principal Investigator: Prof. Volker Erdmann, Free University of Berlin, Germany
Ribonucleic acid (RNA) molecules have diverse biological roles, which include carrying genetic information or amino acids to the ribosomes during protein synthesis and participating as constituents of the ribosomes as they carry out biological functions. Also, RNA molecules may exhibit enzymatic activities. Because of the large mass of its molecules, it has been extremely difficult to crystallise RNA molecules on Earth.
Results:
Of the five crystallisation experiments performed, three
yielded crystals.
The crystals obtained were larger in size and more numerous than
those obtained in simultaneous ground-control experiments. The
largest space-grown crystals had a length of 0.7mm. In the
ground-control experiments, only two chambers yielded crystals.
These were smaller in size and less numerous than those grown in
space. The largest crystal had a length of 0.45 mm. All crystals
were analysed by synchroton radiation at the DESY facilities in
Hamburg, Germany, six days after landing. Both space and ground-
control crystals exhibited a resolution of 13 Å.
Principal Investigator: Dr Richard Giegé, CNRS, Strasbourg, France
Investigators want both to continue and expand the IML-2 crystallisation studies on thermophilic aspartyl-tRNA synthetase and to crystallise the plant sweetening protein, thaumatin. While both proteins are biochemically stable and are purified easily, they also have significant structural and behavioural differences; therefore, they make interesting subjects for comparative crystallography studies. In addition, thaumatin tastes extremely sweet when consumed by humans. Since it appears to be non-toxic, non-carcinogenic, and low in calories, it may be a strong substitute for common table sugar.
Preliminary Conclusions
The quality of the space crystals
obtained in this study is compared directly to those grown under
ground conditions at the same time. Space and Earth-grown
crystals were prepared from the same protein preparation and have
undergone the same manipulation, so that the relative comparisons
between crystals grown in space and on Earth can be significant.
Thaumatin crystals grown in the DIA reactors in space were observed to grow much bigger with significantly less nucleation than the corresponding ground controls. This observation is most striking and is statistically significant, since this phenomenon has been observed in all the reactors containing thaumatin.
The diffraction limits of the crystals grown in space were about the same as those grown on Earth; however, the mosaicity of space-grown crystals was less than that of the ground controls.
Very small crystals were obtained for AspRS, but these crystals were not big enough for analysis. Most reactors containing this enzyme did not crystallise. Investigators speculate that the flight delay may have contributed to the denaturation of the protein, thus hindering its normal crystal growth.
Overall, results indicate a significant difference between quality and size of thaumatin crystals that are grown on Earth and those that are grown in space.
Principal Investigator: Prof. Joseph Martial, University of Liège, Belgium
The long-term goal of investigators is to design a tridimensional 'scaffold' onto which binding and/or active sites for selected peptide sequences could be engineered, eventually producing receptor antagonists that could be used as therapeutic agents for the treatment or prevention of disease. Before this can be accomplished, more information is needed concerning the rules governing protein folding and structure stabilisation. Resolution of the three-dimensional structure of crystals of the synthetic protein, octarellin, may provide these data.
Preliminary Conclusions:
Crystals of Thermotoga TIM suitable for
X-ray data collection were obtained in space, and a clear
difference in the form of these crystals was evident when
compared to the Earth-grown crystals. The effect of microgravity
on the crystal morphology has yet to be investigated.
Crystallisation of Turnip Yellow Mosaic Virus, Tomato Aspermy
Virus, Satellite Panicum Mosaic Virus, Canavalin, Beef Liver
Principal Investigator: Prof. Alexander McPherson, University of California, Riverside, California
Canavalin, catalase, and concanavalin B are being studied to determine the effects of microgravity on protein crystal growth by evaluation of the size, habit, quality, defects, and diffraction properties, including the resolution limit and mosaic spread of the crystals. Three very large proteins, Satellite Panicum Mosaic Virus (with a diameter of 170 Å) along with Turnip Yellow Mosaic Virus and Tomato Aspermy Virus (with diameters of approximately 280 Å) are being studied to verify the theory that the impact of altered transport properties in microgravity should be magnified in proportion to the decreased diffusivity of such large molecules.
Results:
Overall, the USML-2 mission was disappointing for this
investigation, with few crystals grown at all. Only thaumatin
grew high-quality crystals with sizes as large or larger than
those grown on Earth. The quality of the thaumatin crystals was
excellent in all cases, except those where the crystals grew with
faces against walls. These showed extensive striations. One
thaumatin crystal grew from the wall from the point of its
tetragonal face and was safely returned to UCR with no evidence
of perturbation or damage. The thaumatin crystals increased in
size with increasing protein concentrations and diffracted
strongly to the maximum resolution that our data collection
system could achieve, approximately 1.5 Å.
Preliminary Conclusions
The APCF system worked well for the
thaumatin crystals, so it cannot be faulted on fundamental
design. The launch delay was probably detrimental to the validity
of the samples, and the long waiting period before launch
probably degraded the experiment. For future investigations, very
high protein concentrations and lower precipitant concentrations
should be used, as these conditions produced the best-quality
crystals. Crystals grown in the APCF cells were not damaged
physically or perturbed by re-entry and unloading.
Principal Investigator: Prof. Wolfram Sänger, Free University of Berlin, Germany
Protein complexes Photosystem I and Photosystem II are responsible for the primary conversion of visible light into chemical energy in water-oxidising photosynthesis. The objective of this experiment is to elucidate the complete arrangement of chlorophyll molecules, which perform this conversion process in the most efficient way.
Results:
The results of the experiment are very encouraging. On
Earth, the largest of the hexagonal rod-like crystals grew on the
dialysis membrane and was 2 mm long and 0.5 mm Ø (volume of 0.4
mm³). In space, the crystals grow to 4 mm long and 1.5 mm Ø (volume
of 7 mm³). A temperature of 20°C was required for technical
reasons, but the optimum temperature for growth of PSI is
4°C. The diffraction quality of the crystals decays with
time and is worse at 20°C than at 4°C. In spite of
this, the crystals still diffracted to 3.8 Å, and the
mosaic spread reduced slightly to approximately 0.7°. A data
set was collected and is currently being evaluated.
Principal Investigator: Prof. Lennart Sjölin, University of Göteborg, Sweden
One of the more important components of the debate over the need for a convection-free environment for enhanced crystal quality is the comprehensive analysis of the X-ray data from space-grown crystals and from controls, using a variety of statistical techniques to analyse multiple sets of data. By collecting numerous X-ray data sets from each crystal batch, parameters describing crystal perfection can be studied through a statistical comparison of the crystals grown in space to controls grown on Earth. Various hypotheses can thereby be accepted or rejected with a predetermined statistical significance.
Results:
The reactors were inspected immediately after the
flight, and small, irregular crystals were found in three of the
four reactors. These crystals were of poorer quality than other
crystals grown on Earth using regular laboratory equipment. X-ray
analysis was not feasible on these crystals. Instead, some effort
has been spent on understanding why the crystallisation
experiment in space evidently gave crystals of poorer quality
than similar crystallisation experiments in the laboratory.
Principal Investigator: Prof. Gottfried Wagner, University of Giessen, Germany
Bacteriorhodopsin converts light energy to voltages in the membrane of photoenergetic microorganisms that are chemically and genetically distinct from bacteria and higher living organisms. Resolution of the three-dimensional structure of this protein will help scientists understand the mechanisms used to convert light energy to energy for growth.
Results:
In a new experiment protocol, first used under
microgravity conditions during the USML-2 mission, both the
compact alignment of the crystalline filaments of BR and the
increase in crystal size in microgravity, as reported earlier
[Wagner, G. (1994) ESA Journal 18, 25-32], were greatly improved
and resulted in a considerable increase in diffraction power.
Close to the micellar consolution boundary, the molecular rods
of BR were tightly packed together, and the crystal morphology
exhibited smooth surfaces and sharp edges in cubic or needle-
shaped habits of up to 1 mm in length.
Preliminary Conclusions
The new, good-quality BR crystals, grown
in microgravity and on the ground, combined with excellent
synchrotron facilities, allow data collection of the BR crystals
that were shown recently to diffract to a resolution limit of up
to 3.8 Å.
Figure 2: Crystals obtained during USML-2 flight
Principal Investigator: Dr Wolfgang Weber, University of Hamburg, Germany
The receptor for the epidermal growth factor is increasing in its importance as a prognostic factor for a series of human malignancies. Knowledge of the three-dimensional structure of this receptor would open the possibility of tailoring appropriate drugs for the treatment of numerous types of tumors. At the present time, however, the crystal structure of only one hormone receptor (growth hormone) and none of the growth factor receptors have been solved.
Results:
Diffraction data from three crystals were collected
using the synchrotron beam line BW6 at the DORIS storage ring,
DESY Hamburg. The storage ring operated in the main user mode
with 4.5 GeV and up to 100 mA. Images were recorded on the 300-mm
MAR Image Plate scanner at 4°C and at room temperature.
Exposure times were in the range of 3 to 5 minutes for 1.5 degree
rotation using a wavelength of 0.96 Å. The crystal-to-plate
distance was set to 600 mm. The diffraction of all crystals
analysed was comparable: maximum resolution was 6 Å with
a remarkably high quality of spots. The space group P2(sub 1)2(sub1)2
could be evaluated using the DENZO processing package.
Principal Investigator: Prof. Lode Wyns, Free University of Brussels, Belgium
Clarification of the structure and mode of action of the CcdB protein may lead to the design of new antibiotics and anti- tumoral drugs. Specifically, crystal quality needs to be improved and a systematic twinning problem solved. In addition, researchers want to crystallise two specific serine-to-cysteine mutants (Ser74Cys and Ser94Cys), which have not produced crystals large enough for data collection.
Results:
Wild-type CcdB: The ground-control reactor did not
provide any crystals in either the HD or FID reactors. The team
had been unable to obtain any CcdB crystals in an FID ground
setup. The major problem was the appearance of precipitation
immediately after activation of the reactor. In contrast, under
the microgravity conditions of the USML-2 mission, CcdB crystals
were obtained in both the HD and FID reactors. During
investigation of the crystals after the flight, it was found that
twinning was still present, although single crystals were
obtained in the FID reactor. Mutant CcdB: In the ground-control
experiment, small needle-shaped crystals were obtained in the HD,
as well as in the salt chamber of the FID reactor. In the space
HD reactor, the same amount of crystals was obtained but they
were smaller in size. No crystals were obtained in the space FID
reactor; however, postflight activation of this reactor resulted
in a crystallisation (in the salt chamber) comparable to the
ground control.
Principal Investigator: Prof. Ada Yonath, Max-Planck Laboratory for Ribosomal Structure, Hamburg, Germany
Ribosomes are responsible for the translation of the genetic code to proteins. While they are the only organelles in living cells to have been crystallised, most of the Earth-grown crystals are very thin and crack upon handling, causing severe difficulties in data collection and evaluation. The growth chambers of the APCF are almost tailor-made for growing this type of protein crystal, and it may therefore be possible to grow crystals of improved internal order, morphology, size, and mechanical properties. The facility may also allow scientists to control specific properties of the crystal's structure and form.
Results:
Almost every droplet yielded crystals even without
seeding, which is a crucial requirement for the growth of quality
crystals on Earth. Of special importance is the morphology of the
crystals. Although still too small for X-ray crystallography, a
few crystals grown in space are of somewhat better proportions
than those grown on Earth and have a more isotropic shape,
indicating the potential of microgravity. In addition, almost all
crystals grown in space are rather round, a property never
observed on Earth. It is noteworthy that most of the ribosomal
crystals did not break upon return to Earth. This is most
important for this experiment, since the crystals are much more
fragile and delicate than those of average size proteins.
Principal Investigator: Prof. Adriana Zagari, University of Naples, Italy
Alcohol dehydrogenase (ADH) is an enzyme that occurs in large amounts in the livers of mammals, where it plays an important role in several physiological functions, including the breakdown of alcohol. Mammalian ADH is unstable at high temperatures or in the presence of organic solvents, properties that limit its biotechnological application to the synthesis of organic compounds. ADH from Sulfolobus solfataricus, a bacterium that thrives at high temperatures, has greater thermal stability, however, and is scarcely affected by the presence of organic solvents. Given these properties, the enzyme is a good candidate for industrial applications.
Results:
Typically, crystallisation trials using a 6-12 mg/mL
solution of the enzyme in Tris.HCl buffer (0.05-0.15 M), pH 8.4,
NADH 1 mM, with 2-methyl-2, 4-pentane-diol, PMD, as precipitating
agent in the concentration range 44-50% (v/v), produce crystals
large enough to diffract to 3.0 Å resolution. Usually,
crystals grow in size from 400 to 800 mm, within 14 to 30 days.
The launch was delayed by 23 days. The reactors were not refilled
because the protein is usually very stable, and a batch-dependent
variation of the optimal crystallisation conditions is usually
observed. Space experiments have been carried out changing
protein concentration from 8 to 10 mg/mL and MPD concentration
from 46 to 48% (v/v). SsADH crystals were obtained in only two
duplicates out of six reactors, where the protein and the
precipitant concentrations were highest; this was true for both
ground and space experiments. The average size was about 100 mm.
The crystal habit also presented some irregularities. Despite the
small size, two of these crystals were mounted in a Lindmann
capillary and were exposed to the synchrotron radiation at DESY.
The diffraction power was very poor, preventing any further
analysis. The protein, although very stable, occasionally may
undergo slight degradation after a long time. This might have
occurred during the long delay before the launch, resulting in
small-sized crystals.
ESA Microgravity News Vol. 9 No. 1