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Scientists use X-ray crystallography to view molecules that are too small to be seen under a microscope; but this requires crystallizing them, which is difficult to do on Earth. Observing crystallized proteins allows scientists to determine how they are built, which can explain how they work or how other molecules, such as drugs, might interact with them. Growth Rate Dispersion as a Predictive Indicator for Biological Crystal Samples Where Quality Can be Improved with Microgravity Growth (LMM-Biophysics-3) studies ground-based predictions of which crystals benefit from crystallization in microgravity, where Earth's gravity does not interfere with their formation.
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The following content was provided by Eddie H. Snell, Ph.D., and is maintained in a database by the ISS Program Science Office.
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Eddie H. Snell, Ph.D., Hauptman-Woodward Medical Research Institute, Buffalo, NY, United States
Joseph R. Luft, M.S, Hauptman-Woodward Medical Research Institute, Buffalo, NY, United States
NASA Glenn Research Center, Cleveland, OH, United States
ZIN Technologies Incorporated, Cleveland, OH, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
Earth Benefits, Scientific Discovery
ISS Expedition Duration
March 2016 - February 2017; March 2017
- September 2017
Biological macromolecules make up the machinery, the instruction set, and the scaffold of life. They are smaller than the wavelength of visible light, thus sophisticated techniques are needed to visualize them, and through this process, understand how life works. A structural understanding helps to discern mechanisms; once researchers better understand how the machinery works, it is possible to aid, or more commonly impede that machinery, through appropriate pharmaceutical design. The principal means to visualize the structure of these macromolecules is X-ray crystallography, a process that measures the diffraction of X-rays from ordered crystals to calculate the atomic structure. The importance of this process is exemplified in over 12 Nobel Prizes awarded to discoveries made from biological structures derived by X-ray crystallography.
A limiting element to the process is the availability of high-quality, well-diffracting crystals. Quality is defined as a crystal that provides X-ray diffraction data of sufficient completeness and detail (resolution) to see the structure, and hence understand the biology of the system. Biological macromolecules often take many days to grow crystals. Growth in reduced acceleration (commonly termed microgravity) on an orbiting spacecraft extends the physical quality of macromolecular crystals through a reduction in the mosaic spread (caused by slight misalignments of the molecules in the crystal lattice and used as a measure of long-range order), and an increase in crystal volume. Resolution (a measure of short-range order) is not directly affected by microgravity, but can still benefit with the correct experimental design to exploit the demonstrated improvements in long-range order. Microgravity growth can yield an improved quality crystal; however, not all samples improve from microgravity growth.
If it is possible to predict which samples could be improved by crystal growth in microgravity, then the true potential of this medium could be exploited in an efficient manner. Our experiment aims to enable this.
It is known from small molecule studies that a reduction in growth rate dispersion is correlated with an increase in crystal quality. Results from this investigation team show that biological crystals that improve in microgravity show more growth rate dispersion than those that do not. Similarly it has been observed that crystals resulting from growth in microgravity are more uniform in size. It can be postulated that from a simple understanding of the nucleation and growth processes, the growth rate dispersion is reduced in microgravity due to the diffusive, rather than convective, flow regime. This hypothesis is based on these observations, and the theory is that the presence of growth rate dispersion in macromolecular crystals grown on the ground is an indicator of crystals that can be improved when grown in microgravity. The growth rate dispersion of crystals grown on the ground and in microgravity is monitored to determine if there is a correlation between the physical qualities of the resulting crystals with those measurements. It is also proposed to use molecular biology techniques to alter the crystallization contacts, and shift the growth rate dispersion properties of a single protein from low to high, to test this predictive hypothesis. Finally it is proposed that this study be extended to a selection of good and poorly diffracting crystals on the ground, and confirm that those displaying high-growth rate dispersion on the ground are those that are improved on orbit, and generate improved structural data when their quality is exploited.
This experiment uses lysozyme from the T4 bacteriophage. Unlike chicken egg white lysozyme, T4 lysozyme is more representative of biological macromolecules in general. It has been well studied, and extensively mutated to understand folding and other biological processes. T4 lysozyme is produced in the laboratory. Different protein constructs of T4 lysozyme are generated by mutagenesis of the DNA from which the protein is expressed. The mutations produced by this research group are designed to influence the crystallization contacts, and to subtly alter surface areas that may influence protein-protein interactions. These mutations are incorporated into the protein using a Polymerase Chain Reaction (PCR)-based technique with high-fidelity DNA polymerase used to amplify the DNA containing the desired sequence. The original, non-mutated DNA is removed by enzyme digestion. The modified plasmid containing the new DNA sequence is then transformed into bacteria for propagation. This straightforward technique is in routine use in our laboratory and requires the use of standard PCR equipment.
Before crystallization screening, the mutants are characterized with Dynamic Light Scattering, and then the second virial coefficient, B22, measured using Small-angle X-ray scattering (SAXS) techniques with different biochemical conditions. Positive B22 values indicate repulsive intermolecular interactions, while negative values reflect attractive interactions. B22 values in the range from -1x10-4 to -8x10-4 mol*ml g-2, known as the ‘crystallization slot’, have slightly attractive intermolecular interactions and been correlated with crystallization. The aim is to produce subtle shifts in the B22 that change the overall attractiveness of the protein, and thereby alter the growth rate dispersion in a measurable way. Crystallization screening takes place in the research team’s high-throughput crystallization screening laboratory. Conditions are optimized and crystals are grown in multiple replicates of conditions using the batch method, the closest method to mimic that being developed for the Advanced Colloids Experiment (ACE) microscope module.
Growth rate dispersion is measured for different constructs on the ground using a time-lapse microscope with low magnification, so that multiple crystals can be measured over time. The growth rate is measured by an increase in the maximum and minimum linear dimensions, and the total area of the crystal. The former is indicative of anisotropy in the growth process, while the latter is an overall approximation taking into account volume (but not measuring the third dimension). Similar measurements take place in microgravity with the ACE-M apparatus while ground-control experiments use a simple video microscope setup. The growth spread coefficient is calculated by dividing the standard deviation of growth rate by the average growth rate. For insulin the calculation of the growth spread coefficient is 0.28, and for xylose isomerase a growth spread coefficient of 0.14. Insulin has shown improvement in microgravity from this research group’s studies and others, xylose isomerase has not (again from this group’s studies but not reported in the literature due to a null result). A question to be addressed is the quantitative assessment of the growth rate coefficient with respect to improvement, keeping in mind the acceleration environment, i.e. at what value crystals would still be improved given the predicted acceleration level the experiment (a measure of crew activity, distance from center of gravity etc.).
This research group has an expertise in the X-ray analysis of macromolecular crystals, having developed many of the physical characterization methods, and being involved in structural studies. This group has an ongoing program of beamtime at Stanford Synchrotron Radiation Laboratory (SSRL), using this facility for X-ray analysis. Crystals are extracted from the apparatus and initially mounted in quartz glass capillaries. A helical scan procedure, coupled with a Pilatus detector, is used to record a rapid continuous data set, minimizing radiation damage while maximizing the signal due to the reduced mosaicity. The beam is defocused to be as parallel as possible, again maximizing the signal-to-noise. Room-temperature data collection, addressing radiation damage, has the potential for observing biological features that are otherwise masked by traditional cryocooling techniques. This group also employs these cryocooling techniques which while diminishing the physical crystal quality, minimize any radiation damage that may not be overcome by the ambient approach.
Growth rate dispersion is well modeled in the small molecule field, and some studies have extended this to proteins. This research group seeks to build on this coupling the solution measurements, B22, with acceleration conditions (measured during the microgravity conditions) using well developed fluid dynamics techniques to build a model of the crystallization process on orbit. If growth rate dispersion is identified as a predictor of improved quality from microgravity, then candidates that can benefit from the environment can be identified. This research group has time-resolved images of crystallization screening experiments on over 14,000 different proteins. Approximately 50% of these result in crystals, frequently with multiple crystals in the well. Not all of these have crystals in the initial, or even second image, but it can be conservatively estimated that ~3,500 will have crystals appearing at 1 week (based on day to day observation of these results). Each of these proteins usually produces crystals in multiple chemical cocktails (1536 in total). This gives a low fidelity, but unique data set to identify the relative occurrence of growth rate dispersion, a statically valid analysis to identify those samples that might benefit from future flights.
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Using the three-dimensional structure of proteins, scientists can determine how they function and how they are involved in disease. Some proteins benefit from being crystallized in microgravity, where they can grow larger and with fewer imperfections. Access to crystals grown on the International Space Station could improve research for a wide range of diseases, as well as microgravity-related problems such as radiation damage, bone loss and muscle atrophy. This investigation identifies which proteins would benefit from crystallization in space.
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X-ray crystallography is the main method scientists use to study the molecular structures of biology, but it is difficult to crystallize proteins, with only a poor success rate even in the best laboratories. This investigation uses ground-based observations to predict which proteins would benefit from crystallization in microgravity, improving the information that may be obtained from them.
Operational Requirements and Protocols
Sample Storage: The samples are biological proteins contained within a buffer solution, and a precipitant to reduce the solubility of that protein, and cause crystallization under conditions pre-determined on the ground. For the first experiments, the samples are the same protein with one or more amino acids altered on the surface to cause different growth properties. As the protein comes into contact with precipitant, the nucleation and crystallization process is initiated. The process is relatively slow on physical scales, with crystals typically appearing to the eye within an hour to several days, depending on the protein and condition. In the initial iteration, the protein and precipitant solution is prepared on the ground. The protein solution is added to the sample chamber and frozen, followed by the precipitant solution. This keeps both solutions solid so that no crystallization process occurs. The exact minimum temperature is TBD, but it will be higher than -80°C. In this case the facility is transferred to the ISS under frozen conditions, and the experiment activated by thawing either in the Light Microscopy Module (LMM), or outside of it, before transferring the facility to the LMM.
Experimental Chambers: The experimental chambers are where the protein and precipitant are allowed to interact. The experimental objective is to compare crystal quality improvement for those samples that do and do not show growth rate dispersion reduction in microgravity. Ideally two experiments should be conducted side by side, and the ability to duplicate that or study four proteins is desired. While multiple crystals are likely to result in each chamber to ensure statistical validity for observation (and later ground characterization using X-ray methods), six chambers for each sample are required. They could be part of the same fluid path if later injection of fluids becomes possible.
The resulting crystals are non-opaque and can be imaged from a single side using epi illumination. Previous experience with this suggests a cell with a transparent top and black back face. Illumination from behind is used in the laboratory, and could also be adopted with sufficient control on the light and camera gain, and appropriate adjustments for different objectives as necessary. The chamber should have a viewing area that does not cause significant optical distortion and can be completely imaged by the LMM. Crystals are three dimensional. The sample chamber should be large enough to accommodate several crystals up to 1 mm in each dimension.
The crystals are required for X-ray analysis on the return to Earth. The chamber material and solution cause absorption of X-rays at the energy used (typically ~12 keV) so the crystals will need to be extracted from the growth cells. This can be done by opening or cutting the experimental chamber, and in the latter case they should be regarded as disposable elements of the facility.
Experiment initiation Sample cells are frozen on the ground, such that the two solutions, protein and precipitant, are solid. The facility is brought to the ISS and stored frozen until the experiment is to be activated. Activation takes place by thawing the solutions by placing them at the planned observation temperature
Experiment observation: The sample chambers are examined under low magnification (2.5 x) after filling to ensure that they have been completely filled, no air bubbles are present, and to establish a baseline for the start of the experiment. The observation does not have to be instantaneous, as there is a lag time between experiment initiation and observation for the first samples. This lag time is on a protein by protein basis but could be from a few minutes to a few days and is TBD for each protein construct.
A 10X objective is used to image the cells to identify crystal nucleation and locate initial crystals. The first crystals should be a few microns in size.
Crystals are observed as they grow. The 10X objective is used to keep the entire crystal in the field of view. The crystals grow rapidly at first (over the first hours to days), slowing as protein surrounding them is used up, and diffusive transport begins to dominate. Eventual size can reach 1 mm across or more.
Periodically the entire cell/growth chamber is imaged to determine if new crystals have appeared. If new crystals are found these are added to the observation list.
Epi illumination is suitable to be used to ensure a consistent illumination of growing crystals
Subsequent observations are centered on nucleating crystals, and occur automatically unless commanded to change from the ground (i.e. if a crystal moves out of the initial position). This is unlikely once a crystal settles on the sample chamber face.
Observations of each sample cell occur every TBD (based on experiments with a ground unit) minutes during the initial crystal growth stage.
Observation frequency is gradually reduced as determined by experiments to be performed before flights.
Observations continue until growth has slowed to such a point that no change in dimension is detected over a 48 hour period. Ground experiments set a boundary time on this.
The sample cells are imaged as late as possible before return to Earth, so that images recorded on their return can be correlated with those on orbit.
Experiment Control: Initial observations are monitored from the ground to locate the initial crystal positions. Positions are noted, and these positions are used to periodically image the crystals. At a frequency to be determined, the whole growth cell/chamber is reimaged to check for additional crystals. Ground intervention supports in identifying any more crystals, and adding these to the observation list.
If a crystal is discovered to have moved out of frame, the whole growth cell/chamber is reimaged to check for additional crystals. Ground intervention supports in re-identifying crystals, and correcting the observation list.
The experiment requires the ability to move microscope position, and focus, to capture z slices of images across the whole depth of the cell (dependent on optics depth of field). It requires re-defining observation positions and frequency during the experiment.
Environmental Control and Monitoring: Acceleration data is recorded within the LMM to identify deviations of at least 10 micro-g residual acceleration over frequencies of at least 0.1 Hz to 10 Hz. If the LMM is hard attached to the rest of the ISS, calculations of the acceleration at the LMM based on recording elsewhere are acceptable. This is used to correlate any deviations in growth rate with the acceleration environment.
Temperature affects protein solubility, and therefore growth rate. Temperature is maintained at the sample chambers under a specific temperature condition ranging from 12-24°C at +/- 1°C, the desired temperature TBD. Temperature data is recorded at, or close to, the sample chambers at a minimum of 10 minute intervals, with an accuracy of 0.1°C to identify any deviations from the ideal temperature and correlate this with experimental observations.
The experiment should be conducted during a period that minimizes low frequency g-jitter, i.e. avoiding planned reboosts or docking.
The growth chambers/sample cells are stored at the final growth temperature before return to the ground, such that they remain within +/- 1°C of that temperature (TBD). Note, this could be the actual ISS temperature. Sample cells should be returned to earth as soon after the experiment as possible, while maintained within +/- 1°C of the growth temperature. They are not to be frozen after the experiment, as this destroys the crystals.
In-flight Data: Imaging data is required throughout the flight to check that activation has taken place, identify initial crystals, and monitor them as they grow over time.
After experiment activation (thawing), initial images of each sample chamber are required.
During the location of initial crystals, real time or close to real time, imaging is required to confirm crystal location.
Images of the growing crystals are required, but a subsection of those images at a sample rate TBD is permissible.
Information on the microscope position to go with the images is required.
A time stamp with each image received is required.
Post Flight Samples: Part of the experiment is to measure the crystallographic quality of the resulting crystals. For this reason, the sample chambers/growth cells is returned to the ground, and the crystals harvested for X-ray studies.
The following procedural sequence is to be followed to perform the protein crystallization experiments:
A cassette containing 5 to 20 frozen capillaries housed in an observation cassette is removed from a -80°C freezer. There are a suite of Glacier (requires two middeck lockers of space) and Polar (requires one middeck locker space) freezers maintained on the ISS. The capillary cassette is allowed to thaw at ambient ISS temperature and placed in the LMM for crystallization observation (once the frozen solutions containing within the capillaries have thawed the crystallization processes automatically initiated).
The cassette is positioned under a bright field microscope and images recorded at regular intervals (TBD) depending on real-time observation of the crystal growth rate in microgravity for each particular protein construct. It is desirable for the software that controls the XYZ translation stage containing the capillary cassette to automatically reposition itself to crystals photographed during previous sessions (this is important so that at higher magnifications the growth rate of individual crystals can be photographically documented). It is also desirable for the images to be downlinked to the ground-based science team.
After a period of 7 to 21 days the crystals will stop growing (the exact total growth period depends on the crystal growth rate for each protein construct, and the protein concentration in each protein solution). At this point, the cassette can be removed from the LMM and either left at ambient ISS temperature, or if available placed in an incubator maintained at approximately 22°C ± 1°C.
At the conclusion of the mission, the quality of the microgravity-grown crystals is compared to control crystallization experiments performed in 1g. The comparison includes microscopic images of the resultant crystals, X-Ray diffraction analysis comparing the diffraction resolution, diffraction peak widths (mosaicity), and a structural analysis to look at the Bfactors for protein-protein crystallographic contacts for crystals grown in both environments.
Note: All experiments (µg and 1g) for each protein are performed using identical batches of purified protein.
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Native and engineered T4 lysozyme crystals grown using the microbatch method and selected for flight experiments. Figure 1 is the wild type protein and WT signifies a wild type protein with mutations to remove cystines that can cause problems in expression. The letter number letter notation indicates the original amino acid, its numerical position in the sequence and the replacement amino acid in the construct crystallized. Image courtesy of ZIN Technologies.
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