NASA A Researcher’s Guide to International Space Station : Macromolecular Crystal Growth

National Aeronautics and Space Administration
A Researcher’s Guide to:
Macromolecular Crystal Growth
NP-2015-08-027-JSC Macromolecular Crystals-ISS-mini-book-2015.indd 1 10/6/15 2:03 PM

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This International Space Station (ISS) Researcher’s Guide is published by
the NASA ISS Program Science Office.
Authors:
Laurel J. Karr, Ph.D.
Teresa Y. Miller, M.S.
David N. Donovan
Executive Editor: Amelia Rai
Technical Editor: Neesha Hosein
Designer: Cory Duke
Cover and back cover:
a.
Recombinant human insulin crystals grown during STS-95 within the Protein Crystallization Facility
by the temperature induction batch method. (Photos courtesy of the University of Alabama at
Birmingham; publication Smith, Ciszak et al. 1996.)
b.
Enhanced Diffusion-controlled Crystallization Apparatus for Microgravity. (Photo courtesy of NASA’s
Marshall Space Flight Center.)
c.
High Density Protein Crystal Growth. (Photo courtesy of the University of Alabama at Birmingham.)

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CASIS-sponsored experiment flown to the International Space Station on SpaceX 3 and returned to Earth on SpaceX 4.
Large protein crystals are needed for Neutron Diffraction structure determination. Quartz capillaries containing crystals
of inorganic pyrophosphatase grown in space for about six months (A) and crystals grown on Earth (B). Capillaries are 2
mm in diameter. Typical crystals grown in space are shown under polarized light (C; Ng, Baird et al. 2015).
Orbiting the Earth at almost 5 miles per second, a structure exists that is
nearly the size of a football field and weighs almost a million pounds. The
International Space Station (ISS) is a testament to international cooperation
and significant achievements in engineering. Beyond all of this, the ISS is a
truly unique research platform. The possibilities of what can be discovered
by conducting research on the ISS are endless and have the potential to
contribute to the greater good of life on Earth and inspire generations of
researchers to come.
As we increase utilization of ISS as a National Laboratory, now is the time
for investigators to propose new research and to make discoveries
unveiling new knowledge about nature that could not be defined using
traditional approaches on Earth.
The Lab is Open
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1.
Microgravity, or weightlessness, alters many observable phenomena
within the physical and life sciences. Systems and processes affected by
microgravity include surface wetting and interfacial tension, multiphase
flow and heat transfer, multiphase system dynamics, solidification, and
fire phenomena and combustion. Microgravity induces a vast array of
changes in organisms ranging from bacteria to humans, including global
alterations in gene expression and 3-D aggregation of cells into tissue-like
architecture.
2.
Extreme conditions in the ISS environment include exposure to extreme
heat and cold cycling, ultra-vacuum, atomic oxygen, and high-energy
radiation. Testing and qualification of materials exposed to these extreme
conditions have provided data to enable the manufacturing of long-
life, reliable components used on Earth as well as in the world’s most
sophisticated satellite and spacecraft components.
3.
Low-Earth orbit at 51 degrees inclination and at a 90-minute orbit
affords ISS a unique vantage point with an altitude of approximately 240
miles (400 kilometers) and an orbital path over 90 percent of the Earth’s
population. This can provide improved spatial resolution and variable
lighting conditions compared to the sun-synchronous orbits of typical
Earth remote-sensing satellites.
Unique Features of the ISS
Research Environment

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The Lab is Open 3
Unique Features of the ISS Research Environment 5
Why Macromolecular Crystal Growth and Why in Microgravity? 7
Brief History of Macromolecular Crystal Growth in Microgravity 11
Student Involvement 17
What Should Principal Investigators Know About
Conducting Research on ISS 18
Macromolecular Crystal Growth Experiments— Lessons Learned 19
Macromolecular purity, homogeneity and monodispersity 19
Hardware choices 19
Sample volumes required 20
Plan control experiments 20
Crystallization Conditions 20
Multipurpose Facilities Available on the ISS 21
European Drawer Rack (EDR) 21
EXpedite the PRocessing of Experiments for Space Station (EXPRESS) Racks 21
General Laboratory Active Cryogenic ISS Experiment Refrigerator (GLACIER) 22
Gaseous Nitrogen Freezer (GN2) 22
Single-locker Thermal Enclosure System (STES) 23
Commercial Refrigerator Incubator Module – Modified 23
Microgravity Experiment Research Locker Incubator (MERLIN) 24
Polar 24
Kubik 25
Commercial Generic Bioprocessing Apparatus (CGBA) 25
Microgravity Science Glovebox (MSG) 26
Light Microscopy Module (LMM) 26
Nanoracks Microscopes 27
Hardware Designed for Crystallization of Macromolecules 28
Kristallizator (Crystallizer) 28
Image Processing Unit (IPU) 28
Solution Crystallization Observation Facility (SCOF) 29
Protein Crystallization Research Facility (PCRF) 29
NanoRacks-Protein Crystal Growth-1 30
Granada Crystallization Facility (GCF) 30
Protein Crystallization Diagnostics Facility (PCDF) 31
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Enhanced Diffusion-Controlled Crystallization Apparatus for Microgravity (EDCAM) 32
Process for Payload Development 33
Contacts for Macromolecular Crystal Growth Experiments 33
Funding Opportunities 36
Citations 37
Acronyms 45
Table of Contents

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Although macromolecular crystals grown in microgravity consist of proteins, DNA,
RNA and even whole viruses, the vast majority of macromolecular crystals have been
proteins. There are over 100,000 proteins in the human body and an estimated 10
billion throughout the global environment. Proteins serve many functions, including
the maintenance of structure, function and regulation of the body’s tissues and
organs, and provide for catalysis of chemical reactions, cell-to-cell signaling, and
immune responses. To fully understand how they work and how they interact with
each other, it is necessary to determine their 3-D structure. This is most often done
through analysis of X-ray diffraction of quality crystals. A newer method, using
analysis by neutron diffraction, determines the position of hydrogens within a protein
structure and enables more accurate determination of the mechanisms of biochemical
reactions taking place within and between proteins (Blakeley, Langan et al. 2008,
Niimura and Bau 2008). Neutron diffraction requires very large quality crystals,
greater than 1 millimeter3
in volume, in most cases. Fewer than 100 unique neutron
structures of proteins have been reported in the Protein Data Bank, as compared to
over 90,000 X-ray diffraction structures. Figure 1 shows a neutron diffraction-derived
structure of the protein Myoglobin. High-resolution data for X-ray diffraction and
neutron diffraction structure determination requires crystals of high quality with few
defects, and this is often the bottleneck for crystallographers. It is particularly difficult
to grow high-quality crystals of membrane proteins that have the desired qualities, as
evidenced by the fact that only 539 unique structures have been reported since the
first structure was determined in 1985 (Deisenhofer, Epp et al. 1985). It is estimated
that 20-30 percent of all genes in all genomes are integral membrane proteins
(Kahsay, Gao et al. 2005) and that membrane proteins are the targets of over 50
Why Macromolecular
Crystal Growth and Why
in Microgravity?
Figure 1. “Neutron”. (Licensed under Public Domain via
Wikibooks. http://en.wikibooks.org/wiki/File:Neutron.jpg#/
media/File:Neutron.jpg)
Figure 2. Cumulative Unique Membrane Protein Structures.
(http://blanco.biomol.uci.edu/mpstruc/)

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percent of all modern medicinal drugs (Overington, Al-Lazikani et al. 2006). Figure
2 illustrates the progress in membrane structure determination since 1985.
Based on the success of genomic sequencing, in 2000, two institutes of the National
Institutes of Health (the National Institute of General Medical Sciences and the
National Institute of Allergy and Infectious Diseases) collaborated in funding nine
pilot research centers for high-throughput structural determinations. The goal
of these projects was to determine novel structures having less than 30 percent
identity in sequence to proteins whose structures had already been determined
(Norvell and Berg 2007). This five-year effort was renewed and enlarged in 2005.
Similar initiatives were begun in other countries as well. Although many protein
structures have been submitted to the Protein Data Bank, the numbers were not as
high as what was originally anticipated, and one of the bottlenecks, along with the
production of soluble proteins, is successful crystallization (Grabowski, Chruszcz
et al. 2009). A recent set of statistics for one of the most successful centers, the
Northeast Structural Genomics Center, shows that 25,759 proteins have been
cloned, and 6,407 proteins have been purified while only 1,480 of them have
been crystallized successfully (23.3 percent; http://www.nesg.org/statistics.html).
Likewise, the Midwest Center for Structural Genomics has 37,012 active targets
and 3,175 crystals produced (8.5 percent). Of these, 1,843 structures have been
determined (http://www.mcsg.anl.gov/).
Proteins and other macromolecules have been crystallized in microgravity
experiments for over three decades. The first microgravity experiment in protein
crystal growth was in 1981 when Littke conducted a six-minute microgravity
experiment with β-galactosidase on the German TEXUS sounding rocket. Video
from this experiment showed a laminar diffusion process rather than the turbulent
convection that occurs on Earth (Littke and John 1984). Excellent discussions of
the effects of growing macromolecular crystals in microgravity have been published
and in press (Snell and Helliwell 2005, McPherson and DeLucas 2015).
Some characteristics of crystals that are recognized as measurements of quality
include visual perfection and size, resolution limit, I/sigma ratio (in essence
signal-to-noise ratio) and mosaicity. It is believed that factors affecting crystal
growth in microgravity include lack of buoyancy-driven convection and lack of
sedimentation. Pusey et al. illustrated the convection patterns (or growth plumes)
of lysozyme crystals grown in Earth’s gravity (Figure 3; Pusey, Witherow et al.
1988). On Earth, convective flows transport macromolecules to the surface of the
growing crystal, while in microgravity, these buoyancy-driven convective flows are

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not present and the area around the crystal becomes depleted of macromolecules.
Thus, addition of molecules to the growing crystal is governed only by diffusion. It
has been hypothesized that the depleted area around a crystal causes slower growth,
allowing the crystal to form with fewer imperfections and also impedes the addition
of aggregates (because of the slower diffusion of larger molecules; Lin, Rosenberger
et al. 1995; Lin, Petsev et al. 2001). This depletion zone was first visualized by
McPherson and all using Mach-Zhender interferometry on a device called the
Observable Protein Crystal Growth Apparatus (OPCGA; McPherson, J. Malkin
et al. 1999), which was slated for use on the ISS but was canceled following the
Space Shuttle Columbia disaster. More recently, these stable depletion zones around
growing crystals have been visualized and recorded in experiments on the ISS in the
Advanced Protein Growth Facility (APCF; Otalora, Garcia-Ruiz et al. 2002) and in
the Nano Step experiment (Yoshizaki, Tsukamoto et al. 2013).
Many published reports from
microgravity macromolecular growth
experiments have described crystals
having much greater volume than any
grown previously on the ground, which
gave X-ray diffraction data of higher
resolution and I/sigma over the entire
resolution range. Table 1 provides a list
of macromolecules (with references) for
which crystal growth in microgravity
provided significant improvement in
the quality of data over crystals grown
on Earth up to that time. The list is
Figure 3. Schlieren photography shows sequential convective growth plume formation around a lysozyme crystal grown
on Earth (Pusey, Witherow et al. 1988).
A B C D
Figure 4. Comparison of Mosaicity of tetragonal Lysozyme
crystals grown on the ground and in microgravity (Snell,
Weisgerber et al. 1995).

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not all-inclusive, since many experiments flown were for the benefit of commercial
entities, and it is doubtful all of those results have been or will be published. Careful
measurements of the mosaicity of crystals have also shown marked improvement
for microgravity-grown crystals over those grown on Earth. Snell and his colleagues
first reported this in 1995 with tetragonal lysozyme crystals grown on two separate
shuttle missions, in which they demonstrated an improvement by a factor of three
to four over Earth-grown crystals (Snell, Weisgerber et al. 1995; Figure 4). Similar
results comparing microgravity crystals of aminoacyl-tRNA synthetase grown
within dialysis reactors of the European Space Agency’s (ESA’s) Advanced Protein
Crystallization Facility (APCF) on the shuttle STS-78 mission (Ng, Sauter et al.
2002) and with microgravity-grown Insulin crystals on the STS-95 mission (grown
in the commercial Protein Crystallization Facility [PCF]; Borgstahl, Vahedi-Faridi et
al. 2001).
Comparisons of crystals grown in microgravity with those grown on Earth under
the same conditions and in the same equipment is not always the best comparison,
since the best conditions for growth with gravity are often different than the best
conditions in microgravity. Because of this, comparisons for published results were
often between the best conditions seen in microgravity experiments compared with
all of the conditions that had previously been used in Earth laboratories. That there
are so many success stories is fairly remarkable because especially early in the space
shuttle era, the hardware for microgravity experiments had relatively few slots to
screen conditions for optimal crystal growth. So the comparisons were between a few
conditions versus hundreds to thousands of conditions attempted on Earth.
The microgravity conditions aboard the space shuttle were not always optimal,
because of crew activities, minor attitude adjustments and operation of equipment.
Additionally, when accounting for the short time-frames of shuttle missions (usually
7-14 days) and the unforeseen delays in launches, it becomes even more remarkable
that about 40% of the macromolecular crystals grown under microgravity were
of better quality than those grown on Earth, based on the space-grown crystals’
improved x-ray diffraction intensity, resolution and mosaicity (Judge, Snell et al.
2005). The successful samples represented 177 different macromolecules available
for analysis within 63 missions. This group additionally reported that chances
for success were much greater on missions dedicated to providing a microgravity
environment than those that had crystallization experiments as secondary payloads
to other activities, such as satellite launches and retrievals (55 percent success versus
34 percent), and that longer missions trended toward better results, but this was
macromolecule specific. This bodes well for crystallization experiments on the ISS.

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They also found that some macromolecules do better consistently in microgravity
while some do not, an observation which has not yet been adequately explained.
Another review of shuttle experimental results analyzed the number of flights of
macromolecules versus an improvement in diffraction quality and reported about
20 percent of the macromolecules flown obtained the highest diffraction resolution
to date. However, if only macromolecules that were flown more than once were
considered, then the chance of producing better diffracting crystals increased to 35
percent. This illustrates that iterations of crystal growth in microgravity is highly
important (Kundrot, Judge et al. 2001).
Brief History of Macromolecular Crystal Growth in Microgravity
Good reviews of macromolecular crystal growth in microgravity are available, so
this will only be briefly discussed (Lorber 2002; Vergara, Lorber et al. 2003; Judge,
Snell et al. 2005; Snell and Helliwell 2005; McPherson and DeLucas 2015). As
noted above, macromolecular crystal growth in microgravity was first studied by
Littke in 1981 aboard the TEXUS sounding rocket for six minutes in a liquid-
liquid diffusion experiment, showing strictly laminar diffusion patterns (Littke
and John 1984). Macromolecular crystal growth experiments were also included
on some of the unmanned series of Russian Foton satellite missions including
April 1988 (Trakhanov, Grebenko et al. 1991) and 1991, the Foton-3 KASHTAN
experiment (Chayen 1995). In 2007, an ESA-sponsored mission on Foton-M3
provided the first flight of the Granada Crystallization Facility-2 (Gonzalez-
Ramirez, Carrera et al. 2008). Other unmanned experiments included the Swedish
Material Science Experiment Rocket (MASER) flown in 1989 with about seven
minutes of microgravity (Sjölin, Wlodawer et al. 1991), and the China-23, carrying
Crystallization of Organic Substances in Microgravity for Applied Research
(COSIMA-1; Plass-Link 1990).
An experiment based on the TEXUS hardware was flown on STS-9 in 1983 and
grew crystals of lysozyme and β-galactosidase (Littke and John 1986). The Vapor
Diffusion Apparatus (VDA) first flew in 1985 (DeLucas, Suddath et al. 1986).
The design of this hardware was meant to mimic the hanging drop vapor diffusion
experiments most utilized for crystallization on Earth. Many drops were lost during
this experiment, but subsequent refinements were made for later flights. The first
flight with temperature control was STS-26, following the Challenger disaster, also
utilizing the VDA hardware. From this point until about 2004, many shuttle flights
had at least one macromolecular crystal growth experiment, and often two or more.
Also, new designs and methods for crystallization in microgravity came quickly.

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The Protein Crystallization Facility (PCF)
utilizes a large-scale, temperature-based
crystallization method containing 20-500
ml. It first flew on STS-37 in 1991 and has
flown several times since then. Activation is
by temperature ramping, and has been most
used for growth of many crystals having
uniform sizes (Long, DeLucas et al. 1994;
Long, Bishop et al. 1996).
STS-42 (International Microgravity
Laboratory), flown in 1992, was the first
flight to be dedicated to the maintenance of
a microgravity environment. On this flight,
both VDA and the German Cryostat (liquid-
liquid diffusion) hardware were flown. In the
Cryostat, a Satellite Tobacco Mosaic Virus
(STMV) crystal was grown that was 30 times
the volume of any STMV crystal that had
ever been grown on Earth and resulted in a
structure of 1.8 angstroms (Figure 5; Larson,
Day et al. 1998). On STS-50, flown in 1992,
Dr. Larry DeLucas operated a glovebox
experiment enabling iterative experiments
to optimize conditions and practice such
techniques as seeding and crystal mounting
as well as real-time video transfer of data. On
this flight, a malic enzyme crystal was grown, which improved diffraction from 3.2
angstroms to 2.6 angstroms (Figure 6; DeLucas, Long et al. 1994).
Newer designs for crystallization in microgravity began to appear beginning with
STS-57 in 1993. Included in these new designs was ESA’s APCF, which was
temperature controlled, contained 48 individual growth chambers that could
operate either in a batch, dialysis, liquid-liquid, or vapor diffusion mode and could
also provide a video of the growth in 12 of the experiments, as well as a Mach-
Zehnder interferometer available after 1996 (Snyder, Fuhrmann et al. 1991; Bosch,
Lautenschlager et al. 1992; Vergara, Lorber et al. 2003). The capacities of the APCF
were later expanded in 1999 (STS-95) to include the Long Protein-Chamber
Figure 5. Satellite Tobacco Mosaic Virus crystal
grown in Microgravity. (Photo courtesy of Dr. Alexan-
der McPherson, University of California, Irvine)
Figure 6. “Protein Crystal Malic Enzyme.” (Licensed
under Public Domain via Wikimedia Commons. http://
commons.wikimedia.org/wiki/File:Protein_Crystal_Ma-
lic_Enzyme.jpg#/media/File:Protein_Crystal_Ma-
lic_Enzyme.jpg.)

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Free Interface Diffusion reactor utilizing a
counter diffusion technique. In total, the
APCF was flown on six missions including
once on the ISS. An excellent example of
the possibilities of microgravity for the
growth of membrane proteins is shown
in Figure 7. This is the membrane protein
complex Photosystem I, crystallized in
the APCF dialysis mode, which produced
a crystal that was 4 mm in length, and
1.5 mm in diameter, and formed the
basis for an improved crystal structure
(Klukas, Schubert et al. 1999; Fromme and
Grotjohann 2009).
The space shuttle also docked with the
Russian Space Station Mir and carried
macromolecular crystal growth experiments.
In 1989, a vapor diffusion apparatus
was used to crystallize chicken egg white
lysozyme and D-amino transferase,
producing crystals which were larger and
diffracted somewhat better than those grown
in Earth hardware (Stoddard, Strong et
al. 1991). This device used a sitting drop
rather than the hanging drop method used
in the VDA. A more evolved sitting drop
hardware was developed called the Protein
Crystallization Apparatus for Microgravity
(PCAM; Carter, Wright et al. 1999), which
first flew in 1994 as a handheld device and
grew into a hardware that accommodated
many guest investigators and flew 13 times.
Although crystals of many different proteins
were grown in the PCAM, one striking
example is shown in Figure 8 of a manganese
superoxide dismutase crystal that was 80
times larger than any that had grown before
Figure 7. Large, single crystal of Photosystem I, grown
during USML-2 in APCF by dialysis method (Fromme
and Grotjohann 2009).
Figure 8. Examples of microgravity-grown MnSOD
crystals in the PCAM crystallization chamber. (a)
Crystal with dimensions 0.45 x 0.45 x 1.45 mm. The
pink color is due to oxidized manganese in the active
site (not ever seen in the thin crystals grown on Earth).
(b) An example of crystals limited in size to 3 mm in
length by the drop volume (Vahedi-Faridi, Porta et al.
2003).
Figure 5

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on Earth (Vahedi-Faridi,
Porta et al. 2003). The PCAM
could hold 378 samples in
a temperature controlled
locker or 504 samples without
temperature control.
The Gaseous Nitrogen Dewar
(GN2) was first designed for a
flight on Mir, since it required
no temperature control and
no crew time (Koszelak, Leja
et al. 1996). This consisted of
many sealed Tygon tubes with
separately frozen precipitant
and protein solutions. These
were contained in a liquid
nitrogen dewar and as they
gradually thawed, liquid-
liquid diffusion occurred and
the proteins crystallized. This
first experiment contained
183 samples of 19 proteins,
but later refinements
included many more samples,
thereby enabling optimization of growth conditions, and many samples were
devoted to student education projects. This hardware flew many times as the
Enhanced Gaseous Nitrogen (EGN) Dewar. Figure 9 shows pictures of some of
the protein crystals that were grown on the Mir GN2 experiment. The second
hardware designed for Mir is the Diffusion-controlled Crystallization Apparatus for
Microgravity (DCAM), which also required no activation or deactivation by the
crew. The DCAM sample chamber consisted of two cells holding precipitant and
protein, which are separated by a gel plug through which they slowly equilibrate. On
the first flight of DCAM, which occurred on STS-73 as a proof of concept, a crystal
of nucleosome core particle grew that yielded the highest resolution to date (Figure
10 – Carter, Wright et al. 1999; Harp, Hanson et al. 2000). The DCAM hardware
flew seven times, and a second-generation hardware (EDCAM) was designed and
built, but not flown up to this time. A new and larger vapor diffusion apparatus,
Figure 9. X-ray diffraction analysis. Credit: Dr. Alex McPherson, University
of California, Irvine.

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the High Density Protein Crystal
Growth (HDPCG) was designed by the
University of Alabama at Birmingham,
to take the place of the VDA and VDA-
2 (which had a triple-barrel syringe).
This hardware fits into a Microgravity
Experiment Research Locker Incubator
(MERLIN) and can hold up to 1,008
vapor diffusion samples. It flew two
times to the ISS for NASA through 2002
and then again in 2014.
Around 2004, NASA-sponsored
missions in macromolecular crystal
growth were suspended until quite
recently, but ESA, JAXA and Russia
continued the research effort and
kept developing new hardware
and diagnostics. The ESA Granada
Crystallization Facility (GCF) utilizes
a counter-diffusion technique for
crystallization in capillary tubes
(Otalora, Gavira et al. 2009). These
tubes are contained in a Granada
Crystallization Box (GCB), which holds
a maximum of six capillaries, with the
GCF holding 23 GCBs (138 samples
total). This flew on two sortie missions
to the ISS, then JAXA (now NASDA),
used it for nine missions between 2003
and 2009. The NASDA experiments with
GCF were performed in collaboration
with the Russian space agency Roscosmos in the Zvezda service module. NASDA
then developed their own hardware called Protein Crystallization Research Facility
(PCRF), which is located within the Japanese module Kibo (but still launched by
Roscosmos). This new generation of counter-diffusion hardware is said to hold
about 12 times the number of proteins. NASDA has had many success stories
using the GCF and then the PCRF, but one particularly exciting example is the
Figure 10. A. Nucleosome Core Particle crystal, grown
in DCAM on STS-73. B. The structure of the protein was
determined using the crystals grown in space to a 2.5
angstrom resolution (Harp, Hanson et al. 2000), PDB.
http://www.rcsb.org/pdb/explore.do?structureId=1eqz.

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crystallization and structure determination of an inhibitor complexed with the
protein prostaglandin D synthase. This protein is important in allergies and other
inflammations, but is also believed to cause muscle necrosis in Duchenne Muscular
Dystrophy. Results from microgravity experiments produced the highest resolution
structure for this protein. This structure was then used as a template to design a
more potent inhibitor and perhaps a treatment (Aritake, Kado et al. 2006; Mohri,
Aritake et al. 2009; Tanaka, Tsurumura et al. 2011).
The APCF, described above, last flew in 2001 and was replaced with the Protein
Crystallization Diagnostic Facility (PCDF), which is located in the ESA Columbus
Laboratory since 2008. This facility has been used for understanding the
phenomena associated with crystallization processes (Pletser, Bosch et al. 2009;
Patiño-Lopez, Decanniere et al. 2012). The ESA Protein Microscope for the
International Space Station (PromISS) facility was developed for ISS as well. One
operation took place within the U.S. Microgravity Science Glovebox during a sortie
mission on Expedition 12. Complete data sets of 17 crystals of ferritin grown in
PromISS by a counter-diffusion method were compared with complete data sets
of 18 crystals grown under the same conditions on Earth. Statistical analysis was
performed of 63 parameters commonly used as indicators of X-ray data quality, and
it was clearly indicated that the space crystals were of superior quality (Maes, Evrard
et al. 2008).
NASA and the Center for the Advancement of Science in Space (CASIS) have
resumed macromolecular crystal growth experiments within the last few years.
CASIS sponsored a microfluidic experiment using a commercial Plug MakerTM/
CystalCardTM system (Protein BioSolutions), and it was carried to the ISS
aboard the SpaceX Dragon capsule in 2013. This experiment included 25 Crystal
Cards containing about 10,000 individual experiments within two NanoLabs
(NanoRacks). During preparation of the experiment, protein, buffer and precipitant
are mixed in nanoliter quantities in gradient concentrations. Each droplet of
mixture is separated from the next by a biologically inert fluorocarbon, thus giving
10-20 nanoliter microbatch-style crystallization plugs within a small channel.
Sixteen out of 25 cards from microgravity contained crystals while only 12 out of
25 of those on Earth had crystals (Gerdts, Elliott et al. 2008, Carruthers; Gerdts et
al. 2013). This hardware has the advantage of high numbers of screening conditions
of a multitude of proteins using very small volumes. The GCF, described above,
was used again by multiple investigators on the ISS in 2014, sponsored by CASIS.
One of the investigations, led by Joseph Ng, University of Alabama in Huntsville,

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produced unusually large crystals of inorganic pyrophosphatase for neutron
diffraction studies (Ng, Baird et al. 2015).
A NASA-sponsored experiment using a modified version of the HDPCG (described
above) was carried out on the ISS in 2014. The experiment consisted of 360 vapor
diffusion cells each at 4°C and 20°C, 840 liquid-liquid diffusion capillaries at 4°C
and 900 liquid-liquid diffusion capillaries at 20°C. The total number of proteins
flown was 96, at various conditions. Additionally, many of the capillaries contained
experiments from a Science, Technology, Engineering, and Math (also known as
STEM) competition between students from 10 different high schools. Analysis of
results is ongoing.
Student Involvement
As noted above, the most recent high-volume crystallization experiment on ISS
(HDPCG) involved students from 10 different high schools. Over the years that the
EGN Dewar flew, over 50,000 students and 1,090 teachers from 320 schools across
36 states and Puerto Rico had direct involvement with macromolecular crystal
growth through learning curriculums. Moreover, 420 of these students plus 260
of their teachers from 125 schools in 10 states participated in the flight program
including flight sample-loading workshops and launch activities. These efforts as
well as other high school programs, such as those sponsored by the Keck Center for
Molecular Structure at California State University and the Lind laboratory at the
University of Toledo, plus workshops sponsored by the American Crystallographic
Association are all designed to promote the enthusiasm of students for science
and technology, and perhaps to inspire the next generation of crystallographers
(Kantardjieff, Lind et al. 2010).

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Supporting research in science and technology is an important part of NASA’s
overall mission. NASA solicits research through the release of NASA Research
Announcements (NRA), which cover a wide range of scientific disciplines. All NRA
solicitations are facilitated through the Web-based NASA Solicitation and Proposal
Integrated Review and Evaluation System (NSPIRES; http://nspires.nasaprs.com/
external/). Registering with NSPIRES allows investigators to stay informed of newly
released NRAs and enables submission of proposals. NSPIRES supports the entire
lifecycle of NASA research solicitations and awards, from the release of new research
calls through the peer review and selection process.
In planning the scope of their proposal, investigators should be aware of available
resources and the general direction guiding NASA research selection. NASA places
high priority on recommendations from the 2011 National Research Council’s
NRC Decadal Survey, which placed emphasis on hypothesis-driven spaceflight
research. In addition, principal investigators (PIs) should be aware that spaceflight
experiments may be limited by a combination of power, crew time or volume
constraints. Launch and/or landing scrubs are not uncommon, and alternative
implementation scenarios should be considered in order to reduce the risk from
these scrubs. Preliminary investigations using ground-based simulators may be
necessary to optimize procedures before spaceflight. Also, many experiments require
unique hardware to meet the needs of the spaceflight experiment. To understand
previous spaceflight studies, prospective PIs should familiarize themselves with the
NASA ISS Program Science Office database, which discusses research previously
conducted on the ISS, including that of the International Partners. A detailed
catalog of previous, current and proposed experiments, facilities, and results,
including investigator information, research summaries, operations, hardware
information, and related publications is available at www.nasa.gov/iss-science
through the NASA ISS Program Office. Additionally, details pertaining to research
previously supported by the Space Life and Physical Sciences Research and
Applications Division of NASA’s Human Exploration and Operations Mission
Directorate can be located in the Space Life Physical Sciences Research and
Applications Division Task Book in a searchable online database format at: https://
taskbook.nasaprs.com/Publication/welcome.cfm.
What Should Principal
Investigators Know About
Conducting Research
on ISS?

19
When planning macromolecular crystal growth experiments bound for the ISS,
there are some lessons learned from previous missions.
Macromolecule purity, homogeneity and monodispersity
Since flight experiments require so much in the way of time, energy, paper
work, and expense, it is a necessity to spend the extra effort on making sure
one’s favorite macromolecule(s) are as pure, homogeneous, and monodisperse as
possible. Monodispersity can be measured using light, X-ray or neutron scattering
procedures.
Hardware choices
As noted above, the best conditions for growing crystals on the ground are not
necessarily the best conditions in a microgravity environment. After determining
the best conditions obtained on Earth, this should be used as a starting point for
bracketing conditions in microgravity. In general, nucleation and crystal growth is
slower in microgravity than it is on Earth.
It is necessary to know the limitations of the macromolecule and its crystals as well.
Some of the hardware described in this document requires freezing of proteins
and then thawing prior to crystallization. This method can be quite good if the
macromolecule is not degraded by freezing, since launches are sometimes delayed
and frozen samples will not need to be re-loaded prior to launch. Additionally, for
very long flights, samples can be thawed at an appropriate time point for optimal
crystal growth, since some crystals will degrade over time. If possible, it would
be good practice to try more than one method of crystallization, such as vapor
diffusion and liquid-liquid diffusion.
Another consideration is temperature control. If the macromolecule is stable over
a wide range of temperatures, then this will not be of consequence. If, however,
it is temperature sensitive or a temperature gradient is the method of choice for
crystallization, then it will be necessary to use hardware that is carefully temperature
controlled. Although not reported frequently, it is also possible in hardware where
there is a liquid-air interface, such as vapor diffusion, that the crystal may be
subjected to damage that is due to vibration effects or re-entry.
Macromolecular Crystal
Growth Experiments –
Lessons Learned

20
Sample volumes required
Enough sample volume for re-load if a launch gets delayed is highly desirable
(unless frozen samples are used). One also needs to have enough for control
experiments on Earth using equivalent hardware and sample conditions. Also, using
the same macromolecule batch for all needs (flight samples, reloads and controls) is
much better if this can be accomplished, even if small batches have to be pooled to
obtain one large batch. It may be desirable to use hardware that can accommodate
a smaller sample if the macromolecule is very difficult to purify in large quantities,
and therefore very expensive. However, it is possible that a very small sample in a
vapor diffusion apparatus could evaporate during the mission, and if one desires a
very large crystal for neutron diffraction, the size of the crystal can only get as large
as there are macromolecules available to fill it.
Plan control experiments
In many cases, it is best to start control experiments a few days to a week after
activation of the flight experiment, so that it is possible to follow closely the
conditions to which the flight samples are subjected. There are a number of steps in
the flight schedule over which the investigator has no control:
• From loading to launch.
• Transfer of experiment to ISS.
• Activation of experiment.
• Deactivation of experiment.
• Return flight to Earth.
Crystallization conditions
All chemicals that are flown on the ISS must be analyzed for toxicity by the
Toxicology group at NASA’s Johnson Space Center. This normally takes a while
for the group to go through the entire list, so it is best to determine the best
crystallizing conditions as soon as possible. One must bear in mind that chemicals
that are too toxic, even in small quantities, may need to have additional levels of
containment and thus may affect which hardware can be utilized.

21
European Drawer Rack (EDR):
EDR supports seven Experiment
Modules (EMs), each with
independent cooling power and data
communications as well as vacuum,
venting and nitrogen supply, if
required.
EXpedite the PRocessing of
Experiments for Space Station
(EXPRESS) Racks:
EXPRESS Racks is a multipurpose
payload rack system that provides
structural interfaces, power, data
cooling, water, and other items
needed to operate experiments in
space.
Multipurpose Facilities
Available on the ISS
The European Drawer Rack, installed in the Columbus
laboratory. Image was taken during Expedition 16.
Crew member Naoko Yamazaki works to transfer
EXpedite the PRocessing of Experiments
to Space Station Rack 7 from the Multi-Purpose
Logistics Module during STS-131/Expedition 23 Joint
Docked Ops.

22
General Laboratory Active Cryogenic
ISS Experiment Refrigerator (GLACIER):
GLACIER provides a double middeck-
locker-size freezer/refrigerator for a
variety of experiments that require
temperatures ranging from +4°C (39°F)
and -160°C (-301°F). The GLACIER
is compatible with the EXPRESS rack.
It is part of the cold-stowage fleet of
hardware that includes the Minus
Eighty Degree Laboratory Freezer for
ISS and the MERLIN. The GLACIER
incorporates a cold volume sample
storage area of 23.1 cm (10.75 in.) x
27.94 cm (11.00 in.) x 41.91 cm (16.5
in.). It is capable of supporting 10 kg
(22 lb) of experiment samples and has
an internal cold volume of 20 L. The
GLACIER can maintain a temperature
of -160°C (-256°F) for 6 to 8 hours
without power if it has been operating
at -160°C (-301°F) prior to the power
outage.
Gaseous Nitrogen Freezer (GN2):
NASA’s Kennedy Space Center Gaseous
Nitrogen Freezer (GN2) is a passive
freezer container (requires no power). It
is designed to hold samples at cryogenic
temperatures (-196 °C) for between
21 and 35 days, depending on the
flight configuration and use scenario.
The GN2 can be used to transport
frozen samples to and from orbit and
is certified to fly on the International
Space Station.
The sample area inside the internal
tank can hold up to four cylinders
6.0 in. long and 3.7 in. in outer
diameter. In addition, one of the freezer
compartments will be filled preflight
with an insert of the same absorbent
material to increase the thermal mass
of the system. The additional insert is
wrapped with cotton cloth to contain
any particulate.
View of the general Laboratory Active Cryogenic ISS
Experiment Refrigerator within EXpedite the PRocessing
of Experiments to Space Station Rack 6 in the U.S.
Destiny Laboratory during Expedition 18.
NASA’s Kennedy Space Center (KSC) Gaseous Nitrogen
Freezer with lid removed. (Image is courtesy of KSC.)

23
Single-locker Thermal Enclosure
System (STES):
The STES is a single-locker equivalent,
thermally controlled experiment
module capable of operating as either
a refrigerator with a minimum set
point temperature as low as 4.0°C,
or as an incubator with a maximum
set point temperature of 40.0°C. The
STES can maintain a constant set
point temperature, or it may be pre-
programmed to step through a series of
temperatures. The STES can be operated
remotely when installed in an EXPRESS
rack or manually by the crew through
push buttons and a LCD located on the
front panel.
Commercial Refrigerator Incubator
Module – Modified:
The Commercial Refrigerator
Incubator Module - Modified
(CRIM-M) is a single middeck locker
equivalent thermal incubator used for
investigations requiring thermal control
between 4 and 40°C. The CRIM-M
provides thermal performance by power
only and is designed to communicate
with the EXPRESS racks for remote
operations. The internal compartment
provides a 28-V power receptacle and
can control temperature to within
0.5°C. The internal payload volume
dimensions are 17.3 cm x 25.9 cm x
41.9 cm with a maximum allowable
experiment weight of 11.25 kg.
Close-up of the Single-locker Thermal Enclosure
System in Express Rack 4 aboard ISS, during
Expedition 7. The Commercial Refrigerator Incubator Module –
Modified (CRIM-M) is a single, middeck-locker equiva-
lent thermal incubator for payloads requiring thermal
control between 4 and 40°C. Image is courtesy of CBSE
Engineering Division.

24
Microgravity Experiment Research
Locker Incubator (MERLIN):
Microgravity Experiment Research
Locker Incubator (MERLIN) provides a
single, middeck, locker-sized EXPRESS
Rack compatible freezer/refrigerator or
incubator that can be used for a variety
of experiments. Temperature range for
MERLIN is -20°C (-4°F) to + 48.5°C
(+119°F).
Polar:
Polar is a cold stowage managed
facility that provides transport and
storage of science samples at cryogenic
temperatures (-80°C) to and from ISS.
Polar operates on 75 W supplied power
and uses air cooling as its heat-rejection
method. Polar can accommodate up
to 12.75 liters of sample volume and
20 lbm including sample support
equipment.
View of Microgravity Experiment Research Locker/
INcubator on the forward middeck on Space Shuttle
Endeavour. Photo was taken during STS-123 / Expedition
16 joint operations.
Polar Flight Assembly CBSE-F10120-1. Image provided
by the University of Alabama at Birmingham Center for
Biophysical Sciences and Engineering.

25
Kubik:
Kubik consists of a small controlled-
temperature volume, which functions
both as an incubator and cooler (6°C
to 38°C temperature range). Self-
contained automatic experiments,
including crystallization experiments,
seeds, cells, and small animals, are
performed using power provided by
the facility. A centrifuge insert permits
simultaneous 1-g control samples to run
with microgravity samples. There are
no data or command communication
possibilities between the experiments
and Kubik.
Commercial Generic Bioprocessing
Apparatus (CGBA):
The CGBA provides programmable,
accurate temperature control for
applications ranging from cold stowage
to customizable incubation. It provides
automated processing for biological
experiments. The CBGA is designed to
be installed in the EXPRESS rack for
in-orbit operation.
Cosmonaut Salizhan S. Sharipov pictured with the
Kubik incubator aboard the International Space Station.
(Image is courtesy of ESA.)
Photograph of Commercial Generic Bioprocessing
Apparatus during Increment 33 showing open
containment volume and sample canisters. (Image
courtesy of NASA.)

26
Microgravity Science Glovebox (MSG):
The Microgravity Science Glovebox
(MSG) facility on International Space
Station has a large front window
and built-in gloves, creating a sealed
environment to contain liquids and
particles in microgravity for science
and technology experiments. More
than 30 investigations have used the
versatile Glovebox, everything from
material science to life sciences. Ports
are equipped with rugged, sealed gloves
that can be removed when contaminants
are not present, and video and data
downlinks allow experiments to be
controlled from the ground. Researchers
also use MSG to test small parts of
larger investigations and try out new
equipment in microgravity.
Light Microscopy Module (LMM):
Light Microscopy Module (LMM) is
housed within and used in conjunction
with the glovebox in the Fluids
Integrated Rack. Images provided by
the LMM can provide data to scientists
and engineers to help understand the
forces that control the organization
and dynamics of matter at microscopic
scales. The LMM microscope is capable
of using most standard Leica objectives.
The present in-orbit compliment
includes: 2.5x,10x, 20x, 40x, 50x, 63x,
63x oil, and 100x oil objectives. New
or different objectives may also be
flown as needed. The LMM contains
a digital black-and-white, low-noise
scientific camera. 3-D confocal (point
illumination) upgrades are scheduled for
2015-2016.
Expedition 8 Commander and Science Officer Michael
Foale conducts an inspection of the Microgravity
Science Glovebox.
Light Microscopy Module. Image courtesy of NASA’s
Glenn Research Center.

27
Nanoracks Microscopes:
The NanoRacks Microscopes facility
includes three commercial off-the-shelf
optical and reflective microscopes. They
utilize plug and play USB technology
and allow crew members to analyze and
digitally transfer images of ISS in-orbit
samples.
NanoRacks Microscope-3 is an off-the-shelf USB
microscope. Image is courtesy of NanoRacks LLC.

28
Hardware Designed
for Crystallization of
Macromolecules
Kristallizator (Crystallizer):
Kristallizator (Crystallizer) allows the
growth of large protein crystals in
orbit and allows better determination
of the 3-D structure of the crystals,
with applications in applied biology,
medicine, and pharmacology. It is
operated in the CRYOGEM-03 cooler.
Image Processing Unit (IPU):
The Image Processing Unit (IPU) is
a JAXA subrack facility that receives,
records and downlinks experiment
image data for experiment processing.
The IPU is housed in the Ryutai
(fluid) experiment rack with the Fluid
Physics Experiment Facility, Solution
Crystallization Observation Facility
(SCOF), and PCRF.
Kristallizator (Crystallizer). (Image courtesy of the Russian
Federal Space Agency.)
Image Processing Unit. (Image is courtesy of JAXA.)

29
Solution Crystallization Observation
Facility (SCOF):
SCOF is a JAXA subrack facility, located
in the Ryutai (fluid) Rack, which will
investigate morphology and growth of
crystals. The SCOF is equipped with
several microscopes to simultaneously
measure changes in morphology
and growth conditions (temperature
and concentration) of crystals. The
SCOF has an amplitude modulation
microscope and is equipped with two
wavelength interference microscopes
to simultaneously measure changes in
morphology and growth conditions.
Protein Crystallization Research
Facility (PCRF):
The PCRF is a JAXA subrack facility,
located in the Ryutai (fluid) Rack,
which will investigate protein crystal
growth in microgravity. The PCRF can
accommodate six cell cartridges. Each
cell cartridge can accommodate a motor
drive and Peltier elements, from which
activation and termination timing, as
well as temperature profiles, can be
freely designed by the investigator.
An experimental profile appropriate
for each protein can be established.
A CCD camera enables real-time
monitoring of crystal growth. PCRF
Peltier elements installed to cartridges
provide temperature profiles suitable for
target proteins from 0 to 35°C. PCRF
can accommodate six cell cartridges
containing 10 to 16 wells per cartridge
that can hold 10 to 500 microliters per
well. The following methods will be
used to create crystals: Vapor Diffusion;
Batch; Membrane and Liquid-liquid
Diffusion.
Solution Crystallization Observation Facility. Image
courtesy of JAXA.
Astronaut Tim Kopra, Expedition 20 flight engineer,
works at the Protein Crystallization Research Facility
in the Kibo laboratory of the International Space Station.

30
NanoRacks-Protein Crystal Growth-1:
NanoRacks-Protein Crystal Growth-1
(NanoRacks-PCG-1) is a proprietary
protein crystal growth experiment
that utilizes state-of-the-art, on-the-
ground PCG procedures and hardware.
NanoRacks-PCG-1 uses different PCG
solutions in small crystal slides to grow
protein crystals in microgravity. The
slides are launched frozen, thawed in
orbit to allow crystal growth, examined
while in orbit and then returned.
Granada Crystallization Facility (GCF):
The GCF is a multiuser facility designed
to conduct crystallization experiments
of biological macromolecules in
microgravity using a counter diffusion
technique inside capillaries. The
capillaries are enclosed within Granada
Crystallization Boxes (GCBs). It
does not require crew time during
operations and is a passive device (no
electrical power necessary). GCF can
hold 300 crystallization experiments.
It works under diffusion-controlled
mass transport, and the technique
automatically “searches” for the optimal
crystallization conditions.
NanoRacks-Protein Crystal Growth-1 (NanoRacks-
PCG-1) is a proprietary protein crystal growth housed
inside NanoRacks Module-19. Image is courtesy of Carl
W. Carruthers, Jr.
View of the Granada Crystallisation Facility. (Photo
courtesy of ESA.)

31
Protein Crystallization Diagnostics
Facility (PCDF):
The Protein Crystallization Diagnostics
Facility (PCDF) is a multi-user
facility for the investigation of protein
crystal growth and other biological
macromolecules under microgravity.
Crystallization experiments using
the dialysis or the batch method can
be performed. PCDF is designed for
accommodation in the EDR on the ISS.
The facility possesses diagnostic tools
(microscope, optics, interferometers,
video camera) that provide in-depth
knowledge and understanding of
protein crystal growth processes under
microgravity.
High Density Protein Crystal Growth
(HDPCG):
HDPCG holds 1,008 vapor diffusion
samples or a mix of vapor diffusion
and liquid-liquid diffusion samples.
HDPCG is housed in MERLIN for
active thermal control (+4°C to +48°C).
It is suitable for crystallization of a
large variety of aqueous and membrane
proteins involved in critical biological
processes and others that play key roles
in infectious and chronic diseases. One
advantage of this hardware design is the
ability to delay activation of the vapor
diffusion process until the hardware
reaches orbit. This ensures that the
crystallization process is not initiated on
the Launchpad.
The Protein Crystallization Diagnostics Facility.
High Density Protein Crystal Growth. (Image courtesy of
the University of Alabama at Birmingham.)

32
Enhanced Diffusion-Controlled Crystallization Apparatus for Microgravity (EDCAM):
Production of aqueous and membrane protein crystals with improved size and
perfection using liquid/liquid diffusion and dialysis growth methods in support of
structure determination by x-ray and neutron crystallography. Counter-diffusion cells
can be individually programmed to control rate of approach to super-saturation over
periods from several days to months. EDCAM can be flown in thermal carrier or in
passive stowage depending on target investigations. EDCAM is self-activating with
no crew interaction and contains eight Counter-Diffusion Cells per cylinder and 11
cylinders per thermal carrier. Counter-Diffusion Cells could be used for a variety
of experiments including chemical, colloidal, gelation, cell culture additives and/
or fixation studies. The cylinder can be transferred to the Glovebox and the internal
experiments removed for manipulation, activation or viewing.
The Enhanced Diffusion-Controlled Crystallization
Apparatus for Microgravity Image Courtesy of NASA’s
Marshall Space Flight Center.

33
Following selection of an experiment for spaceflight, the PI will work with a
payload integrator or hardware developer to define the most suitable hardware,
and determine if hardware needs to be created or modified. The research team
in combination with payload integrations will establish the specific laboratory
requirements needed to support the experiment. Through these collaborative
efforts, concerns such as crew procedures and crew training, the need for spare parts
and/or contingencies involving hardware, and stowage requirements of the samples
will be addressed and resolved. It is highly recommended that the PI perform
a series of investigations using the identical hardware and under configuration
and control conditions similar to those anticipated in flight prior to the launch.
This will prevent unforeseen issues with the hardware and allow specific mission
constraints to be defined and mitigated prior to the experiment implementation
once aboard the ISS. It is also within this time frame that the science team needs
to characterize the details involved with their synchronous ground controls. The
PI’s team should also have finalized all post-landing procedures, including crystal
storage and transport, and data acquisition prior to the launch.
Another option to flying one’s experiments is through the CASIS (http://www.
iss-casis.org). CASIS is a nonprofit organization tasked by the U.S. Congress and
NASA with promoting and enabling research on ISS. CASIS can be used for all
stages of payload development and can match PIs with implementation partners
(Table 2) who can provide heritage hardware or new flight packages.
Contacts for Macromolecular Crystal Growth Experiments:
CASIS:
Michael S. Roberts, Ph.D. mroberts@iss-casis.org
michael.s.roberts@nasa.gov
Jonathan Volk, Ph.D. jvolk@iss-casis.org
NASA/Space Life Physical Sciences Division:
Francis P. Chiaramonte, Ph.D. francis.p.chiaramonte@nasa.gov
Process for Payload
Development

34
Macromolecule PDB
Identifier
Apparatus Method Data
Purine nucleoside phosphorylase IULA VDA VD (Ealick, Babu et al. 1991)
Interferon γ 1HIG VDA VD (Ealick, Cook et al. 1991)
Human Serum Albumin 1UOR VDA VD (He and Carter 1992)
Phospho carrier protein FAB
complex
1JEL VDA VD (Prasad, Sharma et al. 1993)
Factor D 1DSU VDA VD (Narayana, Carson et al. 1994)
Fkbp12 (immunosuppressant
binding protein)
1FKK VDA VD (Wilson,Yamashita et al. 1995)
Rec. Human Insulin with
phenolic inhibitor
1BEN PCF Temp (Smith, Ciszak et al. 1996)
Antithrombin III 2ANT PCAM VD (Skinner,Abrahams et al.1997)
Satellite tobacco mosaic virus 1A34 CRYOSTAT FID (Larson, Day et al. 1998)
Bacteriophage Lambda
Lysozyme
1AM7 PCAM VD (Evrard, Fastrez et al. 1998)
Eco R1 Endonuclease 1CKQ/
1CL8
PCAM VD (Carter, Wright et al. 1999)
EF-Hand parvalbumin 2PVB PCAM VD (Declercq, Evrard et al. 1999)
Hen Egg White Lysozyme 1BWJ APCF DIA (Dong, Boggon et al. 1999)
Catalase 4BLC EGN FID (Ko, Day et al. 1999)
Photosystem I 1C51 APCF DIA (Klukas, Schubert et al. 1999)
Collagenase 2HLC APCF VD (Broutin-L'Hermite, Ries-Kautt
et al. 2000)
Nucleosome Core Particle 1EQZ DCAM DIA (Harp, Hanson et al. 2000)
Canavalin 1DGW APCF FID (Ko, Day et al. 2000)
Monoclinic Egg White
Lysozyme(neutron diffraction)
n/a DCAM DIA (Ho, Declercq et al. 2001)
Lysozyme 1IEE APCF FID (Sauter, Otalora et al. 2001)
Proteinase K (serine protease) 1IC6 VDA VD (Betzel, Gourinath et al. 2001)
Human Bence-Jones protein 1LGV LMA VD (Alvarado, DeWitt et al. 2001)
[(Pro-Pro-Gly)10]3 Collagen-
like polypeptide
1K6F APCF DIA (Berisio,Vitagliano et al. 2002)
Mistletoe lectin 1M2T HDPCG VD (Krauspenhaar, Rypniewski et
al. 2002)
Alcohol dehydrogenase 1JVB APCF DIA (Esposito, Sica et al. 2002)
NAD synthetase 1KQP VDA VD (Symersky,Devedjiev et al.2002)
Aspartyl-tRNA synthetase 1L0W APCF DIA (Ng, Sauter et al. 2002)
Apocrustacyanin C1 1OBQ APCF VD (Habash, Boggon et al. 2003)
Myoglobin 1NAZ HDPCG VD (Miele, Federici et al. 2003)
T6 Human insulin 1MSO PCF Temp (Smith, Pangborn et al. 2003)
Table 1. Macromolecules whose structures were solved by crystals grown in microgravity, or whose resolution significantly improved.

35
Company Contact Information
The Aerospace Corporation www.aero.org
Astrium North America www.astrium-na.com
Astrotech Corporation www.astrotechcorp.com
Aurora Flight Sciences www.aurora.aero
Bionetics Corporation www.bionetics.com
Bioserve www.colorado.edu/engineering/BioServe
Boeing www.boeing.com
CSS-Dynamac www.css.dynamac.com
Hamilton Sundstrand www.hamiltonsundstrand.com
Jamss America www.jamssamerica.com
Kentucky Space, LLC www.kentuckyspace.com
MDA www.mdacorporation.com
MEI Technologies www.meitechinc.com
Nanoracks LLC www.nanoracks.com
Orbital Technologies Corporation www.orbitec.com
Paragon TEC www.paragontec.net
Qinetiq www.qinetiq-na.com
Space Systems Concepts, Inc. www.space-concepts.com
Space Systems Research Corporation www.spacesystemsresearch.com
Tec-Masters, Inc. www.tecmasters.com
Techshot www.techshot.com
Teledyne Brown Engineering, Inc. www.tbe.com
Thales Alenia Space www.thalesgroup.com/space
UAB www.uab.edu/cbse
Wyle Integrated Science and Engineering www.wyle.com
Zin Technologies www.zin-tech.com
Table 2. Implementation Partners for Flight Experiments on the ISS.

36
There are various avenues that can result in funding for research to be conducted
on the ISS, and the source of funding often dictates the availability of launch
opportunities. Generally, funding for macromolecular crystal growth-related
research is awarded through NASA-sponsored NRA’s, ISS National Laboratory
awards through other government agencies, private commercial enterprise,
nonprofit organizations, and research awards sponsored by the ISS International
Partners. An investigator wanting to fly just a few proteins or other macromolecules
should initiate conversations with the points of contact for individual flight
investigations. If the investigator has chosen a particular hardware he/she would
like to use, then the entity flying that hardware could be contacted (NASA, CASIS,
ESA, NASDA, Roscosmos).
It is not the responsibility of a researcher awarded an ISS flight experiment to fund
costs associated with launch or the ISS laboratory facilities, although industrial
entities may be asked to provide some funding to CASIS for their flights. Greater
detail concerning current funding opportunities for ISS research can be found
through the NASA ISS research website: http://www.nasa.gov/mission_pages/
station/research/ops/research_information.html.
The NASA Solicitation and Proposed Integrated Review and Evaluation System
(NSPIRES) can be accessed via http://nspires.nasaprs.com/external/.
Funding Opportunities

37
Alvarado, U. R., C. R. DeWitt, B. B. Shultz, P. A. Ramsland and A. B. Edmundson
(2001). “Crystallization of a human Bence–Jones protein in microgravity using
vapor diffusion in capillaries.” Journal of Crystal Growth 223(3): 407-414.
Aritake, K., Y. Kado, T. Inoue, M. Miyano and Y. Urade (2006). “Structural and
Functional Characterization of HQL-79, an Orally Selective Inhibitor of Human
Hematopoietic Prostaglandin D Synthase.” Journal of Biological Chemistry 281(22):
15277-15286.
Berisio, R., L. Vitagliano, L. Mazzarella and A. Zagari (2002). “Crystal structure of
the collagen triple helix model [(Pro-Pro-Gly)10]3.” Protein Science 11(2): 262-270.
Betzel, C., S. Gourinath, P. Kumar, P. Kaur, M. Perbandt, S. Eschenburg and T. P.
Singh (2001). “Structure of a serine protease proteinase K from Tritirachium album
limber at 0.98 A resolution.” Biochemistry 40(10): 3080-3088.
Blakeley, M. P., P. Langan, N. Niimura and A. Podjarny (2008). “Neutron
crystallography: opportunities, challenges, and limitations.” Current Opinion in
Structural Biology 18(5): 593-600.
Borgstahl, G. E., A. Vahedi-Faridi, J. Lovelace, H. D. Bellamy and E. H. Snell
(2001). “A test of macromolecular crystallization in microgravity: large well ordered
insulin crystals.” Acta Crystallogr D Biol Crystallogr 57(Pt 8): 1204-1207.
Bosch, R., P. Lautenschlager, L. Potthast and J. Stapelmann (1992). “Experiment
equipment for protein crystallization in μg facilities.” Journal of Crystal Growth
122(1-4): 310-316.
Broutin-L’Hermite, I., M. Ries-Kautt and A. Ducruix (2000). “1.7 A X-ray structure of
space-grown collagenase crystals.” Acta Crystallographica Section D 56(3): 376-378.
Carruthers, C. W., C. Gerdts, M. D. Johnson and P. Webb (2013). “A Microfluidic,
High Throughput Protein Crystal Growth Method for Microgravity.” PLoS ONE
8(11, e82298).
Carter, D. C., B. Wright, T. Miller, J. Chapman, P. Twigg, K. Keeling, K. Moody,
M. White, J. Click, J. R. Ruble, J. X. Ho, L. Adcock-Downey, T. Dowling, C.-H.
Chang, P. Ala, J. Rose, B. C. Wang, J.-P. Declercq, C. Evrard, J. Rosenberg, J.-P.
Wery, D. Clawson, M. Wardell, W. Stallings and A. Stevens (1999). “PCAM: a
multi-user facility-based protein crystallization apparatus for microgravity.” Journal
of Crystal Growth 196(2-4): 610-622.
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45
APCF Advanced Protein Crystallization Facility
CGBA Commercial Generic Bioprocessing Apparatus
COSIMA Crystallization of Organic Substances in Microgravity for Applied Research
CRIM-M Commercial Refrigerator Incubator Module – Modified
DCAM Diffusion-controlled Crystallization Apparatus for Microgravity
EDCAM Enhanced Diffusion-Controlled Crystallization Apparatus for Microgravity
EDR European Drawer Rack
EGN Enhanced Gaseous Nitrogen (EGN) Dewar
EM Experiment module
ESA European Space Agency
EXPRESS EXpedite the PRocessing of Experiments to Space Station
GCB Granada Crystallization Boxes
GCF Granada Crystallization Facility
GLACIER General Laboratory Active Cryogenic ISS Experiment Refrigerator
GN2 Gaseous Nitrogen Freezer
GN2 Gaseous Nitrogen-Dewar
HDPCG High Density Protein Crystal Growth
IPU Image Processing Unit
ISS International Space Station
JAXA Japan Aerospace Exploration Agency
LMM Light Microscopy Module
MASER Material Science Experiment Rocket
MERLIN Microgravity Experiment Research Locker Incubator
MSG Microgravity Science Glovebox
NASDA National Space Development Agency of Japan
NIH National Institutes of Health
NSPIRES NASA Solicitation and Proposal Integrated Review and Evaluation System
OPCGA Observable Protein Crystal Growth Apparatus
PCAM Protein Crystallization Apparatus for Microgravity
PCDF Protein Crystallization Diagnostics Facility
PCRF Protein Crystallization Research Facility
PI Principal Investigator
PromISS Protein Microscope for the International Space Station
SCOF Solution Crystallization Observation Facility
STES Single-locker Thermal Enclosure System
STMV Satellite Tobacco Mosaic Virus
VDA Vapor Diffusion Apparatus
Acronyms

46
46
The Complete ISS Researcher’s
Guide Series
1. Acceleration Environment
2. Cellular Biology
3. Combustion Science
4. Earth Observations
5. Fluid Physics
6. Fruit Fly Research
7. Fundamental Physics
8. Human Research
9. Macromolecular Crystal Growth
10. Microbial Research
11. Microgravity Materials Research
12. Plant Science
13. Rodent Research
14. Space Environmental Effects
15. Technology Demonstration

47
47
For more information...
Space Station Science
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Facilities
http://www.nasa.gov/mission_pages/station/research/
facilities_category.html
ISS Interactive Reference Guide:
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Researchers/Opportunities
http://www.nasa.gov/mission_pages/station/research/
ops/research_information.html

48
National Aeronautics and Space Administration
Johnson Space Center
http://www.nasa.gov/centers/johnson
www.nasa.gov
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