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Protein structural changes on a CubeSat under rocket acceleration profile

Protein structural changes on a CubeSat under rocket acceleration profile www.nature.com/npjmgrav BRIEF COMMUNICATION OPEN Protein structural changes on a CubeSat under rocket acceleration profile 1,7 2,7 3 4 5,6✉ Autumn Luna , Jacob Meisel , Kaitlin Hsu , Silvia Russi and Daniel Fernandez Catalyzing life-sustaining reactions, proteins are composed by 20 different amino acids that fold into a compact yet flexible three- dimensional architecture, which dictates what their function(s) might be. Determining the spatial arrangement of the atoms, the protein’s 3D structure, enables key advances in fundamental and applied research. Protein crystallization is a powerful technique to achieve this. Unlike Earth’s crystallization experiments, biomolecular crystallization in space in the absence of gravitational force is actively sought to improve crystal growth techniques. However, the effects of changing gravitational vectors on a protein solution reaching supersaturation remain largely unknown. Here, we have developed a low-cost crystallization cell within a CubeSat payload module to exploit the unique experimental conditions set aboard a sounding rocket. We designed a biaxial gimbal to house the crystallization experiments and take measurements on the protein solution in-flight with a spectrophotometry system. After flight, we used X-ray diffraction analysis to determine that flown protein has a structural rearrangement marked by loss of the protein’s water and sodium in a manner that differs from crystals grown on the ground. We finally show that our gimbal payload module design is a portable experimental setup to take laboratory research investigations into exploratory space flights. npj Microgravity (2020) 6:12 ; https://doi.org/10.1038/s41526-020-0102-3 INTRODUCTION system. First, we built a crystallization cell system based on the well-known bulk dialysis crystallization method employed by the Second to water, a typical human cell is by mass 20% protein of 4 5 1 pioneers of protein crystallography . In a diffusion cell, a which there are ~10 –10 different species . Proteins sustain the concentrated solution of protein is separated by a semipermeable complex life processes of gravity-evolved multicellular organisms membrane from a larger volume of a concentrated solution of and, to a bigger or lesser degree, they can acclimate to changes in 2–5 precipitant. Diffusion will took place through the membrane gravity as several studies have suggested . Reduced gravity retaining molecular species of higher molecular weight with offers boundless opportunities for technological exploration in 6 7 respect to the membrane’s pore size. We used inexpensive acrylic areas such as tissue engineering , biomaterials , energy produc- 8 9 boxes with lid to create the diffusion cell, and to monitor the tion , geological prospection , or for in-space supply manufactur- protein solution, we adapted a circuit with a UV-light-emitting ing . A further technological application, crystallization of 11 12 13–15 diode and a detector (Fig. 1a). This design allows for a space- mineral , inorganic , colloidal , and macromolecular sam- efficient, portable configuration, and a cost-effective means to ples, has been widely explored. perform crystallization experiments. Although crystal growth rate The goal of macromolecular crystallography is to reveal the is marginally important in standard crystallography analysis, it is macromolecule’s three-dimensional (3D) structure at the atom’s however critical for our payload experiment. We chose lysozyme level by using X-ray diffraction on crystals obtained from a variety of as the subject of our study because of its biological relevance and experimental setups . Crystallization in space begun ~40 years ago its widespread use in a laboratory setting. Lysozyme belongs to a with the idea that in microgravity the crystals will grow with major family of enzymes that specifically hydrolyze murein, a enhanced properties that would eventually improve the quality of 17,18 peptidoglycan which is the insoluble polymer that forms the cell the derived X-ray diffraction data . Yet, protein crystallization in wall of bacteria; in fact, in humans, lysozyme is distributed mostly space is not entirely a perturbation-free process as transient in tissues in contact to airborne pathogens, suggesting a accelerations that may affect the crystal growth process originate 22,23 24 19,20 protective role . Adapting a fast crystal-growing procedure , from onboard human activity and/or space mission operations . we obtained a large number of crystals within the very limited time window determined by the flight (~60 s). RESULTS In-solution protein experimental cell design Temperature control, gimbal assembly, and recovery of the experimental cell We sought to understand what these changing acceleration vectors might cause on a supersaturated protein solution when a Then, to successfully recover the protein samples for after-flight space mission begins, upon the rocket’s motor burn, and during analysis, we designed a temperature-controlled (temperature is a spaceflight. To this end, we designed a new biaxial gimbal critical variable for crystal growth) and shock-reducing (to prevent experimental crystallization setup for a 1.5 U CubeSat payload crystal damage) portable container. On launch ground, 1 2 Mechanical Engineering Department, School of Engineering, Stanford University, Stanford, CA 94305, USA. Electrical Engineering Department, School of Engineering, Stanford 3 4 University, Stanford, CA 94305, USA. Biology Department, School of Humanities and Sciences, Stanford University, Stanford, CA 94305, USA. Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratories, Menlo Park, CA 94025, USA. Stanford ChEM-H Macromolecular Structure Knowledge Center (MSKC), Stanford 6 7 University, Stanford, CA 94305, USA. Stanford ChEM-H Institute, Stanford University, Stanford, CA 94305, USA. These authors contributed equally: Autumn Luna, Jacob Meisel. email: danilo@stanford.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; A. Luna et al. Fig. 1 Flowchart for the design of the experimental crystallization cell payload. a Schematic view of the payload crystallization cell principle of operation. The crystallization cell is a 25 mm × 25 mm × 19 mm acrylic box that holds the semipermeable cellulose membrane molded into a conical shape to contain the protein and provide a large contact area to the precipitant solution. The precipitant solution, a mixture of buffer, salt, and the water-soluble polymer PEG, is delivered via the syringe-pump device upon launch to initiate crystallization. A low-cost, single-wavelength UV LED and photodiode was coupled to the experiment to monitor the protein solution. b Testing insulation at simulating high-temperature launch conditions in the laboratory. The CubeSat unit was assembled with ice packs added in the bulkheads and the unit placed in an oven at 50 °C. Inside the unit, temperature reached a low of 22 °C after adding the ice packs, and then it gradually began to rise at a rate of 0.23 °C/min. Due to the temperature never reaching lower than 22 °C, resistive heating was not employed for the launch. c Gimbal interior showing two crystallization cells, deployment syringe, and wiring. An inertial measurement unit was used to measure the orientation and acceleration experienced within the inner gimbal box. A microSD to record experimental data and a printed circuit board assembly around a SAMD21 microcontroller complete the electronics system. To power the system, we used 1100 mAh LiPo batteries and a 3.3 V buck-boost voltage converter. temperatures can reach more than 35 °C and they may descend to the crystallization cell. During rocket’s preparation and gimbal −45 °C at altitude 9100 m. To keep temperature close to the range integration the readings of UV-light intensity transmitted through crystals were obtained in the preliminary laboratory tests, we the protein solution were unchanged, but upon motor burn and resorted to energy-saving, low-weight-practicable resources. injection of the precipitant, the solution was under large Temperature was moderated by polymer ice packs and aerogel acceleration during 6.5 s (Fig. 2a, “Hypergravity”). The sharpest insulation encasing the CubeSat structure, and by placing 12 Ω spike of transmitted UV intensity is observed at the largest resistors adjacent to the crystallization cell for Joule heating. We excursion of the acceleration vectors whose overall magnitude conducted laboratory tests with the assembled payload at a reaches 6.82 g (Fig. 2b). After motor burn, the rocket decelerated temperature close to what was to be expected on the desert from drag for 7.5 s, which generated an apparent freefall for the launch ground and found that the insulation lining prevented crystallization cell (Fig. 2a, “Microgravity”). During this time, the UV rapid shifts in the inside of the module despite external readings show a rapid decrease in intensity, suggesting that the temperature reaching 50 °C simulated in an oven, even after solution transitions to microgravity conditions. After the rocket many hours of high-temperature exposure (Fig. 1b). For tempera- reached peak altitude, a parachute recovery system was ejected, tures under 20 °C we adopted resistive heating; however, as our and the assembly quickly reached terminal velocity (Fig. 2a, flight conditions were towards higher temperatures, it was not “Terminal Velocity”). In this final stage, the crystallization cell needed. Before rocket’s launch, exterior ice packs were placed experienced 1 g for 42 s and showed non-varying UV readings around the payload to minimize heating, but due to delays, the (Fig. 2b, right side). In summary, the flight accelerometer and UV- interior temperature was around 36 °C at time of launch. The light readings indicate marked changes of the directional CubeSat was secured within the rocket for the duration of the acceleration magnitudes on the lysozyme solution as it super- launch. Rocket recovery is composed of two parachute ejection saturated with the precipitant to rapidly form nascent micro- events to reduce landing speed to 8.8 m/s. A 0.76 m diameter crystals. However, it was not possible establishing whether a given spherical parachute and a 1.62 m diameter toroidal parachute are acceleration vector component acted preferentially on any deployed at maximum altitude and 460 m above the ground, particular crystal growth dimension, but rather they have altered respectively. To minimize vibration, we selected a spring-laden, the crystal properties altogether. freely moving gimbal subsystem that levels upon the main system’s movements (Fig. 1c). All the necessary hardware to Protein polymorphism at the atom’s level initiate, monitor, and record the experiment are housed in the To better understand the effect of the rocket’s acceleration profile gimbal, including: the precipitant deployment system, the UV on the dialysis crystallization experiment, we analyzed an in-flight spectrophotometer, the temperature control, the accelerometer, grown crystal and a ground control crystal through X-ray the data acquisition card, electronics, and power supply. The diffraction. Table 1 lists the crystallographic data. Unexpectedly, entire gimbal system fits within a 1.5 U CubeSat frame (Supple- the space-grown and Earth-grown crystals are non-isomorphic: mentary Figs. 1 and 2), is modular and easy-to-integrate into the the lengths of the crystal lattice vectors in the μg lysozyme rocket, and it does not interfere with launch operations. collapse by ~6% while the crystal’s solvent content is ~10% less than the 1 g lysozyme. A survey of publicly available data indicates In-flight protein solution transformations that our μg lysozyme is the smallest among 622 tetragonal The gimbal system was integrated and assembled into the lysozyme structures. These results are not inconsistent with CubeSat module and launched. With the protein already loaded previous observations on the dynamic structural flexibility of a onto the membrane, the precipitant solution was injected upon related phage lysozyme crystallized under a variety of chemical rocket’s ascent. To track the evolution of the protein solution while precipitants , and imply that the protein has replaced water- in flight, we used a light-emitting source at 280 nm that traverses mediated contacts by protein–protein contacts and/or that npj Microgravity (2020) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; A. Luna et al. Fig. 2 Lysozyme solution evolving to crystals and protein structural changes. a Zoom in on flight accelerometer data, and b protein solution UV-light measurements. On rocket’s ascent above the 2.9 g preset acceleration threshold, the precipitant solution is injected into the crystallization cell triggering changes to the protein solution on the other side of the semipermeable membrane. The system is firstly under varying acceleration conditions, then the acceleration decreases sharply to microgravity to finally reach terminal velocity (1 g). c Protein crystal packing environment of lysozyme in the Earth-grown crystal. Six symmetry-related mole- cules are depicted along the crystallographic c axis. Only backbone atoms from N-terminal Lys1 (labeled n) to C-terminal Leu129 (c) are shown as a tube in gray. Interface interactions of the β-sheet 43–46 are in dark gray (b) and of the sodium-bound loop 61–78 (l, in black) are highlighted. The sodium ion (sphere in black) is separated by 14.8 Å from a neighboring molecule ion and the interstices are filled by water molecules (not shown for clarity). d Packing of the space- grown lysozyme crystal. Lysozyme backbone atoms from N-terminal Lys1 (n) to C-terminal Cys127 (c) are shown as a tube in gray. Approximately the same orientation as before. Numbers indicate the positions of amino acids for which coordinates could accurately be determined (positions that could not be traced in the electron density maps include 43–45 of the β-sheet and 67–72 in the long loop 61–78). Two Earth-grown lysozyme molecules (black) were overlaid onto two neighboring space-grown molecules to illustrate that sodium-binding is noncompatible with this dry packing arrangement. molecules on a manner that differs from the well organized 1 g lysozyme. Taken together, these results demonstrate that local changes affecting a small portion (10% of the total amino acid components) in flexible, solvent-exposed areas that mediate intermolecular contacts could have made possible the smaller μg crystal without paying the penalty of de-folding lysozyme. These results suggest that upon launch the protein solution experienced a shock such that by hypergravity solutes of lower molecular weight than the cutoff of the semipermeable mem- brane were passed through to the bulk precipitant solution. We cannot exclude the possibility however that the acceleration vectors acting on the protein molecule might have triggered the conformational change on the flexible, solvent-exposed areas and that the protein remodeled the sodium-binding site, releasing it. Either way, the low-solvent content μg crystal is the smallest among the tetragonal lysozyme crystals with available 3D coordinates. By overall crystallographic statistics our rocket- grown crystal was of lower quality than the ground lysozyme crystal raising the possibility that our design was not a problem- free container for this fragile cargo. This cannot be ascribed only to hardware though as a previous study concluded that dissimilar protein crystallization outcomes aboard a sounding rocket would changed global conformation. Inspection of the 3D structure have been caused by factors related to reentry or by the crystal reveals that despite their different growth regimes the overall growth process itself . To summarize, our gimbal design structure is preserved and is indistinguishable with the canonical rendered an experimental setup for a biophysical experiment lysozyme fold. However, the largest positional changes concen- that, shuttled in a CubeSat frame, showed the altering effects of a trated on two areas that mediate interface contacts with crystal rocket’s acceleration profile on a protein in solution. lattice neighbors: at the β-sheet (residues 42–46) and within the long-coiled loop region (residues 66–73). Interestingly, the sodium-binding site within the coiled loop is well organized in DISCUSSION 1 g lysozyme (Fig. 2c) while it is abrogated in the μg crystal (Fig. We developed a biaxial system to be fitted into a 1.5 U CubeSat as 2d).This finding is in line with a recent study on orthorhombic a biology research experiment payload and adapted a protein lysozyme that shows this sodium-binding site is likely to have a crystallization experiment to span the very short timeframe of the conformational change with loss of the metal ion .In1g rocket’s flight. Crystal growth under rocket’s flight conditions lysozyme, the sodium site is at 14.8 Å of a symmetry-related Na affected the way the protein molecules assembled in the crystal, ion organizing an intermolecular interface thanks to surrounding driving local conformational changes that resulted in the loss of water. Such an arrangement however cannot be possible made in water and protein-bound sodium. The limited timescale of the the μg lysozyme as the smaller lattice vectors would place the + + Na –Na sites closer by 1 Å provoking spatial clashes between flight would however render our system of little utility to most amino acid side chains (compare Fig. 2c, d). Similarly, the β-sheet protein crystallization experiments, even though for difficult changes in the μg crystal to make interfacial contacts to neighbor crystallization problems the simplicity of the method of bulk Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 12 A. Luna et al. diffusion could be used for screening crystallization conditions on Table 1. Data collection and refinement statistics. 28,29 a larger scale. In this work and in previous reports with model proteins, high-g forces produced crystals from or by achieving μg Lysozyme 1 g Lysozyme supersaturated solutions. The goal would be obtaining nuclei or Data collection nascent crystals to be used as seeds for conventional crystal- Beamline SSRL BL12–2 SSRL BL14-1 lization on Earth or under microgravity in the International Space Wavelength (Å) 0.97946 1.19499 Station. A more suitable recovery system for the CubeSat Space group P4 22P4 2 2 3 1 3 1 experimental cell and systematic exploration at high-g on a Cell dimensions variety of precipitants and macromolecules would be needed to a, b, c (Å) 75.97, 75.97, 34.87 78.87, 78.87, 37.15 test the feasibility of the system. Additional application of the α, β, γ (°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 experimental cell on a sounding rocket would be in quickly Unit cell/asymmetric unit vol 201,250/25,156 231,098/28,886 evolving systems such as enzyme kinetics, where these reactions (Å ) occur typically on the order of milliseconds or less and would Mosaicity (°) 0.29 0.15 require tightly controlled, fast solution-mixing, and quick data Wilson B-factor 59.6 20.7 acquisition systems. Interestingly, we have demonstrated here 3 c Matthews coefficient (Å /Da) 1.73 1.99 that rocket’s acceleration readings triggered a pump-controlled Solvent content (%) 29.1 38.3 injection of a chemical reactant that ultimately led to protein Resolution (Å) 53.72 (2.67) 39.43 (1.60) structural changes. Incorporating spectral monitoring and fast- R 0.085 (0.777) 0.045 (0.619) acting syringe pumps our device would promise an easy-to- merge I/σI ratio 11.7 (2.3) 10.9 (1.7) integrate, portable, and low-cost platform for pilot studies destined to better understand fundamental biological process in Completeness (%) 99.6 (99.7) 96.2 (99.6) space activities. Redundancy 6.1 (6.7) 3.2 (3.0) Refinement Resolution (Å) 30.0–2.67 30.0–1.60 METHODS No. reflections/test set 2912/209 14,505/782 i Crystallization reagents and apparatus R /R 24.4/30.6 17.0/22.0 work free Commercially available hen egg white lysozyme lyophilized powder was F − F correlation 0.93 0.97 obs calc from MP Biomedicals, LLC (Solon, OH, USA). Sodium acetate (Mallinckrodt No. atoms Baker, Inc., Paris, KY, USA), sodium chloride (Fisher Scientific, Fair Lawn, NJ, Protein 874 1014 USA), polyethylene glycol 6000 (Sigma-Aldrich, Merck KGaA, Darmstadt, Ligand/ion 1 (chlorine) 5 (1 sodium, 4 chlorine) Germany) were dissolved in ultrapure Milli-Q water (EMD Millipore, Merck Water 6 140 KGaA, Darmstadt, Germany) and the stock solutions filtered through a 0.45-μm syringe filter. Lysozyme working solutions were made by B-factors weighing and dissolving the lysozyme in 50 mM sodium acetate buffer, Protein 76.5 24.8 pH 4.5. Protein was dialyzed/concentrated using a 3 kDa MW cutoff Ligand/ion 65.6 30.1 microcon centrifugal device (EMD Millipore, Merck KGaA, Darmstadt, Water 52.9 38.8 Germany). Protein concentration was measured using a NanoDrop 2000c R.m.s. deviations UV–Visible spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, Bond lengths (Å) 0.011 0.020 USA). The crystallization cell was a 25 mm × 25 mm × 19 mm acrylic box Bond angles (°) 1.44 1.94 purchased from a home goods products store. We chose a regenerated cellulose tubing commonly used for dialysis in the laboratory as a readily Ramachandran statistics available, cheap, and biocompatible medium. We utilized a semipermeable Most favored regions (%) 100 100 cellulose membrane of 7 kDa MW cutoff without any activation as Disallowed regions (%) 0 0 instructed by the manufacturer (Thermo Fisher Scientific, Wilmington, Degree of crystal imperfection, a higher mosaicity contributes to broader DE, USA). Square pieces of membrane of 50 mm × 50 mm were cut and (less sharply defined) diffraction intensity profiles. molded with a machined finger tool before attachment to the container Overall B-factor value, an approximation to the falloff of atomic scattering box. The membrane was molded into a conical shape such that the vertex with resolution. is in contact to the precipitant solution and its external rim provides Ratio of the volume of the asymmetric unit to the molecular weight of all tightening for the lid sealing the chamber. protein molecules in the asymmetric unit. Value in parentheses is for the highest-resolution shell: 2.67–2.82 Å in μg Design of the crystallization cell and gimbal system lysozyme and 1.60–1.64 Å in 1 g lysozyme. We developed our own microfluidic precipitant deployment system, Reliability factor for symmetry-related reflections calculated as: R = merge including a syringe-pump and pinch valve, which a micro servo motor Σ Σj = 1 to N|I − I (j)|/Σ Σj =1toNI (j), where N is the hkl hkl hkl hkl hkl releases upon launch. This syringe-pump design delivers the 2 ml of redundancy of the data. In parentheses, the cumulative value at the precipitant solution to the parallel crystallization cells in one second. A low- highest-resolution shell. cost, single-wavelength UV LED and photodiode spectrophotometer was Ratio of mean intensity to the mean standard deviation of the intensity coupled to the experiment to measure the protein absorbance at 280 nm. over the entire resolution range. The UV photodiode has a 250–310 nm responsivity band. The spectro- Fraction of measured reflections to possible observations at the photometer system was tested in the laboratory to determine the gain and resolution range. absorbance of the components of the crystallization cell. The box material Number of measurements of individual, symmetry unique reflections. is not UV-light-transparent and cutouts were done on the acrylic box and Average deviation between the observed and calculated structure factors lid and sealed with polyethylene film to provide the maximal possible calculated as: R = Σ ||F | − |F ||/Σ |F |, where the F and F work hkl obs calc hkl obs obs calc intensity excursion. At a distance of 25 mm to the photodiode, the UV are the observed and calculated structure factor amplitudes of reflection absorption intensity of the experimental box was decreased by only 1.35% hkl. R is equal to R but for a randomly selected 5.0% (6.3% in μg free factor compared with the intensity measured in its absence. An inertial lysozyme) subset of the total reflections that were held aside throughout measurement unit (Bosch Sensortec BNO055) is used to measure the refinement for cross-validation. orientation and acceleration experienced within the inner gimbal box. A Correlation coefficient between observed and calculated structure factor microSD to record experimental data and a printed circuit board around a amplitudes. According to Procheck for non-proline and non-glycine residues. SAMD21 microcontroller complete the electronics system. Again, for space constraints in the inner gimbal box, we used 1100 mAh, 3.7 V, 18350 LiPo batteries, and a 3.3 V buck-boost voltage converter. To provide thermal npj Microgravity (2020) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA A. Luna et al. control we chose long-lasting ice packs, aerogel fiber insulation, and Joule 6. Pietsch, J. et al. 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Crystallogr. 26, 283–291 (1993). s41526-020-0102-3. 41. Delano, W. Pymol: an open-source molecular graphics tool. CCP4 Newsl. Protein Crystallogr. 40,82–92 (2002). Correspondence and requests for materials should be addressed to D.F. 42. Burley, S. K. et al. RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, bio- Reprints and permission information is available at http://www.nature.com/ technology and energy. Nucleic Acids Res. D1, D464–D474 (2019). reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims ACKNOWLEDGEMENTS in published maps and institutional affiliations. This study was funded by the Stanford Student Space Initiative (SSI) organization. We thank H. Perry at the Stanford Environmental Health & Safety Surplus Chemical Program for some of the reagents used in the study. We apologize to all those investigators whose papers could not be quoted in this article due to space limitations. We thank two anonymous reviewers for their constructive comments. Use Open Access This article is licensed under a Creative Commons of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Attribution 4.0 International License, which permits use, sharing, Laboratory, is supported by the US Department of Energy, Office of Science, Office of adaptation, distribution and reproduction in any medium or format, as long as you give Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural appropriate credit to the original author(s) and the source, provide a link to the Creative Molecular Biology Program is supported by the DOE Office of Biological and Commons license, and indicate if changes were made. The images or other third party Environmental Research, and by the National Institutes of Health, National Institute of material in this article are included in the article’s Creative Commons license, unless General Medical Sciences (including P41GM103393). The contents of this publication indicated otherwise in a credit line to the material. If material is not included in the are solely the responsibility of the authors and do not necessarily represent the article’s Creative Commons license and your intended use is not permitted by statutory official views of NIGMS or NIH. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. AUTHOR CONTRIBUTIONS A.L., J.M., and D.F. designed the study. A.L., J.M., S.R., K.H., and D.F. carried out © The Author(s) 2020 experiments. D.F. contributed reagents/materials/analysis tools. D.F. wrote the paper. All authors discussed findings and commented on the paper. npj Microgravity (2020) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

Protein structural changes on a CubeSat under rocket acceleration profile

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www.nature.com/npjmgrav BRIEF COMMUNICATION OPEN Protein structural changes on a CubeSat under rocket acceleration profile 1,7 2,7 3 4 5,6✉ Autumn Luna , Jacob Meisel , Kaitlin Hsu , Silvia Russi and Daniel Fernandez Catalyzing life-sustaining reactions, proteins are composed by 20 different amino acids that fold into a compact yet flexible three- dimensional architecture, which dictates what their function(s) might be. Determining the spatial arrangement of the atoms, the protein’s 3D structure, enables key advances in fundamental and applied research. Protein crystallization is a powerful technique to achieve this. Unlike Earth’s crystallization experiments, biomolecular crystallization in space in the absence of gravitational force is actively sought to improve crystal growth techniques. However, the effects of changing gravitational vectors on a protein solution reaching supersaturation remain largely unknown. Here, we have developed a low-cost crystallization cell within a CubeSat payload module to exploit the unique experimental conditions set aboard a sounding rocket. We designed a biaxial gimbal to house the crystallization experiments and take measurements on the protein solution in-flight with a spectrophotometry system. After flight, we used X-ray diffraction analysis to determine that flown protein has a structural rearrangement marked by loss of the protein’s water and sodium in a manner that differs from crystals grown on the ground. We finally show that our gimbal payload module design is a portable experimental setup to take laboratory research investigations into exploratory space flights. npj Microgravity (2020) 6:12 ; https://doi.org/10.1038/s41526-020-0102-3 INTRODUCTION system. First, we built a crystallization cell system based on the well-known bulk dialysis crystallization method employed by the Second to water, a typical human cell is by mass 20% protein of 4 5 1 pioneers of protein crystallography . In a diffusion cell, a which there are ~10 –10 different species . Proteins sustain the concentrated solution of protein is separated by a semipermeable complex life processes of gravity-evolved multicellular organisms membrane from a larger volume of a concentrated solution of and, to a bigger or lesser degree, they can acclimate to changes in 2–5 precipitant. Diffusion will took place through the membrane gravity as several studies have suggested . Reduced gravity retaining molecular species of higher molecular weight with offers boundless opportunities for technological exploration in 6 7 respect to the membrane’s pore size. We used inexpensive acrylic areas such as tissue engineering , biomaterials , energy produc- 8 9 boxes with lid to create the diffusion cell, and to monitor the tion , geological prospection , or for in-space supply manufactur- protein solution, we adapted a circuit with a UV-light-emitting ing . A further technological application, crystallization of 11 12 13–15 diode and a detector (Fig. 1a). This design allows for a space- mineral , inorganic , colloidal , and macromolecular sam- efficient, portable configuration, and a cost-effective means to ples, has been widely explored. perform crystallization experiments. Although crystal growth rate The goal of macromolecular crystallography is to reveal the is marginally important in standard crystallography analysis, it is macromolecule’s three-dimensional (3D) structure at the atom’s however critical for our payload experiment. We chose lysozyme level by using X-ray diffraction on crystals obtained from a variety of as the subject of our study because of its biological relevance and experimental setups . Crystallization in space begun ~40 years ago its widespread use in a laboratory setting. Lysozyme belongs to a with the idea that in microgravity the crystals will grow with major family of enzymes that specifically hydrolyze murein, a enhanced properties that would eventually improve the quality of 17,18 peptidoglycan which is the insoluble polymer that forms the cell the derived X-ray diffraction data . Yet, protein crystallization in wall of bacteria; in fact, in humans, lysozyme is distributed mostly space is not entirely a perturbation-free process as transient in tissues in contact to airborne pathogens, suggesting a accelerations that may affect the crystal growth process originate 22,23 24 19,20 protective role . Adapting a fast crystal-growing procedure , from onboard human activity and/or space mission operations . we obtained a large number of crystals within the very limited time window determined by the flight (~60 s). RESULTS In-solution protein experimental cell design Temperature control, gimbal assembly, and recovery of the experimental cell We sought to understand what these changing acceleration vectors might cause on a supersaturated protein solution when a Then, to successfully recover the protein samples for after-flight space mission begins, upon the rocket’s motor burn, and during analysis, we designed a temperature-controlled (temperature is a spaceflight. To this end, we designed a new biaxial gimbal critical variable for crystal growth) and shock-reducing (to prevent experimental crystallization setup for a 1.5 U CubeSat payload crystal damage) portable container. On launch ground, 1 2 Mechanical Engineering Department, School of Engineering, Stanford University, Stanford, CA 94305, USA. Electrical Engineering Department, School of Engineering, Stanford 3 4 University, Stanford, CA 94305, USA. Biology Department, School of Humanities and Sciences, Stanford University, Stanford, CA 94305, USA. Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratories, Menlo Park, CA 94025, USA. Stanford ChEM-H Macromolecular Structure Knowledge Center (MSKC), Stanford 6 7 University, Stanford, CA 94305, USA. Stanford ChEM-H Institute, Stanford University, Stanford, CA 94305, USA. These authors contributed equally: Autumn Luna, Jacob Meisel. email: danilo@stanford.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; A. Luna et al. Fig. 1 Flowchart for the design of the experimental crystallization cell payload. a Schematic view of the payload crystallization cell principle of operation. The crystallization cell is a 25 mm × 25 mm × 19 mm acrylic box that holds the semipermeable cellulose membrane molded into a conical shape to contain the protein and provide a large contact area to the precipitant solution. The precipitant solution, a mixture of buffer, salt, and the water-soluble polymer PEG, is delivered via the syringe-pump device upon launch to initiate crystallization. A low-cost, single-wavelength UV LED and photodiode was coupled to the experiment to monitor the protein solution. b Testing insulation at simulating high-temperature launch conditions in the laboratory. The CubeSat unit was assembled with ice packs added in the bulkheads and the unit placed in an oven at 50 °C. Inside the unit, temperature reached a low of 22 °C after adding the ice packs, and then it gradually began to rise at a rate of 0.23 °C/min. Due to the temperature never reaching lower than 22 °C, resistive heating was not employed for the launch. c Gimbal interior showing two crystallization cells, deployment syringe, and wiring. An inertial measurement unit was used to measure the orientation and acceleration experienced within the inner gimbal box. A microSD to record experimental data and a printed circuit board assembly around a SAMD21 microcontroller complete the electronics system. To power the system, we used 1100 mAh LiPo batteries and a 3.3 V buck-boost voltage converter. temperatures can reach more than 35 °C and they may descend to the crystallization cell. During rocket’s preparation and gimbal −45 °C at altitude 9100 m. To keep temperature close to the range integration the readings of UV-light intensity transmitted through crystals were obtained in the preliminary laboratory tests, we the protein solution were unchanged, but upon motor burn and resorted to energy-saving, low-weight-practicable resources. injection of the precipitant, the solution was under large Temperature was moderated by polymer ice packs and aerogel acceleration during 6.5 s (Fig. 2a, “Hypergravity”). The sharpest insulation encasing the CubeSat structure, and by placing 12 Ω spike of transmitted UV intensity is observed at the largest resistors adjacent to the crystallization cell for Joule heating. We excursion of the acceleration vectors whose overall magnitude conducted laboratory tests with the assembled payload at a reaches 6.82 g (Fig. 2b). After motor burn, the rocket decelerated temperature close to what was to be expected on the desert from drag for 7.5 s, which generated an apparent freefall for the launch ground and found that the insulation lining prevented crystallization cell (Fig. 2a, “Microgravity”). During this time, the UV rapid shifts in the inside of the module despite external readings show a rapid decrease in intensity, suggesting that the temperature reaching 50 °C simulated in an oven, even after solution transitions to microgravity conditions. After the rocket many hours of high-temperature exposure (Fig. 1b). For tempera- reached peak altitude, a parachute recovery system was ejected, tures under 20 °C we adopted resistive heating; however, as our and the assembly quickly reached terminal velocity (Fig. 2a, flight conditions were towards higher temperatures, it was not “Terminal Velocity”). In this final stage, the crystallization cell needed. Before rocket’s launch, exterior ice packs were placed experienced 1 g for 42 s and showed non-varying UV readings around the payload to minimize heating, but due to delays, the (Fig. 2b, right side). In summary, the flight accelerometer and UV- interior temperature was around 36 °C at time of launch. The light readings indicate marked changes of the directional CubeSat was secured within the rocket for the duration of the acceleration magnitudes on the lysozyme solution as it super- launch. Rocket recovery is composed of two parachute ejection saturated with the precipitant to rapidly form nascent micro- events to reduce landing speed to 8.8 m/s. A 0.76 m diameter crystals. However, it was not possible establishing whether a given spherical parachute and a 1.62 m diameter toroidal parachute are acceleration vector component acted preferentially on any deployed at maximum altitude and 460 m above the ground, particular crystal growth dimension, but rather they have altered respectively. To minimize vibration, we selected a spring-laden, the crystal properties altogether. freely moving gimbal subsystem that levels upon the main system’s movements (Fig. 1c). All the necessary hardware to Protein polymorphism at the atom’s level initiate, monitor, and record the experiment are housed in the To better understand the effect of the rocket’s acceleration profile gimbal, including: the precipitant deployment system, the UV on the dialysis crystallization experiment, we analyzed an in-flight spectrophotometer, the temperature control, the accelerometer, grown crystal and a ground control crystal through X-ray the data acquisition card, electronics, and power supply. The diffraction. Table 1 lists the crystallographic data. Unexpectedly, entire gimbal system fits within a 1.5 U CubeSat frame (Supple- the space-grown and Earth-grown crystals are non-isomorphic: mentary Figs. 1 and 2), is modular and easy-to-integrate into the the lengths of the crystal lattice vectors in the μg lysozyme rocket, and it does not interfere with launch operations. collapse by ~6% while the crystal’s solvent content is ~10% less than the 1 g lysozyme. A survey of publicly available data indicates In-flight protein solution transformations that our μg lysozyme is the smallest among 622 tetragonal The gimbal system was integrated and assembled into the lysozyme structures. These results are not inconsistent with CubeSat module and launched. With the protein already loaded previous observations on the dynamic structural flexibility of a onto the membrane, the precipitant solution was injected upon related phage lysozyme crystallized under a variety of chemical rocket’s ascent. To track the evolution of the protein solution while precipitants , and imply that the protein has replaced water- in flight, we used a light-emitting source at 280 nm that traverses mediated contacts by protein–protein contacts and/or that npj Microgravity (2020) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; A. Luna et al. Fig. 2 Lysozyme solution evolving to crystals and protein structural changes. a Zoom in on flight accelerometer data, and b protein solution UV-light measurements. On rocket’s ascent above the 2.9 g preset acceleration threshold, the precipitant solution is injected into the crystallization cell triggering changes to the protein solution on the other side of the semipermeable membrane. The system is firstly under varying acceleration conditions, then the acceleration decreases sharply to microgravity to finally reach terminal velocity (1 g). c Protein crystal packing environment of lysozyme in the Earth-grown crystal. Six symmetry-related mole- cules are depicted along the crystallographic c axis. Only backbone atoms from N-terminal Lys1 (labeled n) to C-terminal Leu129 (c) are shown as a tube in gray. Interface interactions of the β-sheet 43–46 are in dark gray (b) and of the sodium-bound loop 61–78 (l, in black) are highlighted. The sodium ion (sphere in black) is separated by 14.8 Å from a neighboring molecule ion and the interstices are filled by water molecules (not shown for clarity). d Packing of the space- grown lysozyme crystal. Lysozyme backbone atoms from N-terminal Lys1 (n) to C-terminal Cys127 (c) are shown as a tube in gray. Approximately the same orientation as before. Numbers indicate the positions of amino acids for which coordinates could accurately be determined (positions that could not be traced in the electron density maps include 43–45 of the β-sheet and 67–72 in the long loop 61–78). Two Earth-grown lysozyme molecules (black) were overlaid onto two neighboring space-grown molecules to illustrate that sodium-binding is noncompatible with this dry packing arrangement. molecules on a manner that differs from the well organized 1 g lysozyme. Taken together, these results demonstrate that local changes affecting a small portion (10% of the total amino acid components) in flexible, solvent-exposed areas that mediate intermolecular contacts could have made possible the smaller μg crystal without paying the penalty of de-folding lysozyme. These results suggest that upon launch the protein solution experienced a shock such that by hypergravity solutes of lower molecular weight than the cutoff of the semipermeable mem- brane were passed through to the bulk precipitant solution. We cannot exclude the possibility however that the acceleration vectors acting on the protein molecule might have triggered the conformational change on the flexible, solvent-exposed areas and that the protein remodeled the sodium-binding site, releasing it. Either way, the low-solvent content μg crystal is the smallest among the tetragonal lysozyme crystals with available 3D coordinates. By overall crystallographic statistics our rocket- grown crystal was of lower quality than the ground lysozyme crystal raising the possibility that our design was not a problem- free container for this fragile cargo. This cannot be ascribed only to hardware though as a previous study concluded that dissimilar protein crystallization outcomes aboard a sounding rocket would changed global conformation. Inspection of the 3D structure have been caused by factors related to reentry or by the crystal reveals that despite their different growth regimes the overall growth process itself . To summarize, our gimbal design structure is preserved and is indistinguishable with the canonical rendered an experimental setup for a biophysical experiment lysozyme fold. However, the largest positional changes concen- that, shuttled in a CubeSat frame, showed the altering effects of a trated on two areas that mediate interface contacts with crystal rocket’s acceleration profile on a protein in solution. lattice neighbors: at the β-sheet (residues 42–46) and within the long-coiled loop region (residues 66–73). Interestingly, the sodium-binding site within the coiled loop is well organized in DISCUSSION 1 g lysozyme (Fig. 2c) while it is abrogated in the μg crystal (Fig. We developed a biaxial system to be fitted into a 1.5 U CubeSat as 2d).This finding is in line with a recent study on orthorhombic a biology research experiment payload and adapted a protein lysozyme that shows this sodium-binding site is likely to have a crystallization experiment to span the very short timeframe of the conformational change with loss of the metal ion .In1g rocket’s flight. Crystal growth under rocket’s flight conditions lysozyme, the sodium site is at 14.8 Å of a symmetry-related Na affected the way the protein molecules assembled in the crystal, ion organizing an intermolecular interface thanks to surrounding driving local conformational changes that resulted in the loss of water. Such an arrangement however cannot be possible made in water and protein-bound sodium. The limited timescale of the the μg lysozyme as the smaller lattice vectors would place the + + Na –Na sites closer by 1 Å provoking spatial clashes between flight would however render our system of little utility to most amino acid side chains (compare Fig. 2c, d). Similarly, the β-sheet protein crystallization experiments, even though for difficult changes in the μg crystal to make interfacial contacts to neighbor crystallization problems the simplicity of the method of bulk Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 12 A. Luna et al. diffusion could be used for screening crystallization conditions on Table 1. Data collection and refinement statistics. 28,29 a larger scale. In this work and in previous reports with model proteins, high-g forces produced crystals from or by achieving μg Lysozyme 1 g Lysozyme supersaturated solutions. The goal would be obtaining nuclei or Data collection nascent crystals to be used as seeds for conventional crystal- Beamline SSRL BL12–2 SSRL BL14-1 lization on Earth or under microgravity in the International Space Wavelength (Å) 0.97946 1.19499 Station. A more suitable recovery system for the CubeSat Space group P4 22P4 2 2 3 1 3 1 experimental cell and systematic exploration at high-g on a Cell dimensions variety of precipitants and macromolecules would be needed to a, b, c (Å) 75.97, 75.97, 34.87 78.87, 78.87, 37.15 test the feasibility of the system. Additional application of the α, β, γ (°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 experimental cell on a sounding rocket would be in quickly Unit cell/asymmetric unit vol 201,250/25,156 231,098/28,886 evolving systems such as enzyme kinetics, where these reactions (Å ) occur typically on the order of milliseconds or less and would Mosaicity (°) 0.29 0.15 require tightly controlled, fast solution-mixing, and quick data Wilson B-factor 59.6 20.7 acquisition systems. Interestingly, we have demonstrated here 3 c Matthews coefficient (Å /Da) 1.73 1.99 that rocket’s acceleration readings triggered a pump-controlled Solvent content (%) 29.1 38.3 injection of a chemical reactant that ultimately led to protein Resolution (Å) 53.72 (2.67) 39.43 (1.60) structural changes. Incorporating spectral monitoring and fast- R 0.085 (0.777) 0.045 (0.619) acting syringe pumps our device would promise an easy-to- merge I/σI ratio 11.7 (2.3) 10.9 (1.7) integrate, portable, and low-cost platform for pilot studies destined to better understand fundamental biological process in Completeness (%) 99.6 (99.7) 96.2 (99.6) space activities. Redundancy 6.1 (6.7) 3.2 (3.0) Refinement Resolution (Å) 30.0–2.67 30.0–1.60 METHODS No. reflections/test set 2912/209 14,505/782 i Crystallization reagents and apparatus R /R 24.4/30.6 17.0/22.0 work free Commercially available hen egg white lysozyme lyophilized powder was F − F correlation 0.93 0.97 obs calc from MP Biomedicals, LLC (Solon, OH, USA). Sodium acetate (Mallinckrodt No. atoms Baker, Inc., Paris, KY, USA), sodium chloride (Fisher Scientific, Fair Lawn, NJ, Protein 874 1014 USA), polyethylene glycol 6000 (Sigma-Aldrich, Merck KGaA, Darmstadt, Ligand/ion 1 (chlorine) 5 (1 sodium, 4 chlorine) Germany) were dissolved in ultrapure Milli-Q water (EMD Millipore, Merck Water 6 140 KGaA, Darmstadt, Germany) and the stock solutions filtered through a 0.45-μm syringe filter. Lysozyme working solutions were made by B-factors weighing and dissolving the lysozyme in 50 mM sodium acetate buffer, Protein 76.5 24.8 pH 4.5. Protein was dialyzed/concentrated using a 3 kDa MW cutoff Ligand/ion 65.6 30.1 microcon centrifugal device (EMD Millipore, Merck KGaA, Darmstadt, Water 52.9 38.8 Germany). Protein concentration was measured using a NanoDrop 2000c R.m.s. deviations UV–Visible spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, Bond lengths (Å) 0.011 0.020 USA). The crystallization cell was a 25 mm × 25 mm × 19 mm acrylic box Bond angles (°) 1.44 1.94 purchased from a home goods products store. We chose a regenerated cellulose tubing commonly used for dialysis in the laboratory as a readily Ramachandran statistics available, cheap, and biocompatible medium. We utilized a semipermeable Most favored regions (%) 100 100 cellulose membrane of 7 kDa MW cutoff without any activation as Disallowed regions (%) 0 0 instructed by the manufacturer (Thermo Fisher Scientific, Wilmington, Degree of crystal imperfection, a higher mosaicity contributes to broader DE, USA). Square pieces of membrane of 50 mm × 50 mm were cut and (less sharply defined) diffraction intensity profiles. molded with a machined finger tool before attachment to the container Overall B-factor value, an approximation to the falloff of atomic scattering box. The membrane was molded into a conical shape such that the vertex with resolution. is in contact to the precipitant solution and its external rim provides Ratio of the volume of the asymmetric unit to the molecular weight of all tightening for the lid sealing the chamber. protein molecules in the asymmetric unit. Value in parentheses is for the highest-resolution shell: 2.67–2.82 Å in μg Design of the crystallization cell and gimbal system lysozyme and 1.60–1.64 Å in 1 g lysozyme. We developed our own microfluidic precipitant deployment system, Reliability factor for symmetry-related reflections calculated as: R = merge including a syringe-pump and pinch valve, which a micro servo motor Σ Σj = 1 to N|I − I (j)|/Σ Σj =1toNI (j), where N is the hkl hkl hkl hkl hkl releases upon launch. This syringe-pump design delivers the 2 ml of redundancy of the data. In parentheses, the cumulative value at the precipitant solution to the parallel crystallization cells in one second. A low- highest-resolution shell. cost, single-wavelength UV LED and photodiode spectrophotometer was Ratio of mean intensity to the mean standard deviation of the intensity coupled to the experiment to measure the protein absorbance at 280 nm. over the entire resolution range. The UV photodiode has a 250–310 nm responsivity band. The spectro- Fraction of measured reflections to possible observations at the photometer system was tested in the laboratory to determine the gain and resolution range. absorbance of the components of the crystallization cell. The box material Number of measurements of individual, symmetry unique reflections. is not UV-light-transparent and cutouts were done on the acrylic box and Average deviation between the observed and calculated structure factors lid and sealed with polyethylene film to provide the maximal possible calculated as: R = Σ ||F | − |F ||/Σ |F |, where the F and F work hkl obs calc hkl obs obs calc intensity excursion. At a distance of 25 mm to the photodiode, the UV are the observed and calculated structure factor amplitudes of reflection absorption intensity of the experimental box was decreased by only 1.35% hkl. R is equal to R but for a randomly selected 5.0% (6.3% in μg free factor compared with the intensity measured in its absence. An inertial lysozyme) subset of the total reflections that were held aside throughout measurement unit (Bosch Sensortec BNO055) is used to measure the refinement for cross-validation. orientation and acceleration experienced within the inner gimbal box. A Correlation coefficient between observed and calculated structure factor microSD to record experimental data and a printed circuit board around a amplitudes. According to Procheck for non-proline and non-glycine residues. SAMD21 microcontroller complete the electronics system. Again, for space constraints in the inner gimbal box, we used 1100 mAh, 3.7 V, 18350 LiPo batteries, and a 3.3 V buck-boost voltage converter. To provide thermal npj Microgravity (2020) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA A. Luna et al. control we chose long-lasting ice packs, aerogel fiber insulation, and Joule 6. Pietsch, J. et al. Three-dimensional growth of human endothelial cells in an heating. In all, the main power consumption is for heating (57%) and to automated cell culture experiment container during the SpaceX CRS-8 ISS space power the UV LED, sensor, microcontroller, accelerometer, and SD reader mission—the SPHEROIDS project. Biomaterials 124, 126–156 (2017). (33%). The remainder 10% is left as safety margin. To start the 7. Zea, L. et al. Design of a spaceflight biofilm experiment. Acta Astronaut. 148, crystallization experiment, we use linear acceleration data to detect launch 294–300 (2018). and to prevent false-starts from jostling the system during integration, an 8. Brinkert, K. et al. Efficient solar hydrogen generation in microgravity environment. acceleration threshold of 2.9 g for 250 ms was selected. Nat. Commun. 9, 2527 (2018). 9. Asphaug, E. et al. A cubesat centrifuge for long duration milligravity research. Npj Microgravity 3, 16 (2017). 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E. & Suvorova, statistics and validation reports are presented in Table 1. The X-ray E. I. Crystallization of calcium phosphate in microgravity. Adv. Space Res. 16, diffracting power of space-grown and Earth-grown crystals differed 65–68 (1995). qualitatively. The highest dispersion for a space-grown crystal was to a 13. Zhu, J. et al. Crystallization of hard-sphere colloids in microgravity. Nature 387, minimum Bragg spacing of 2.67 Å. The crystal belonged to the 883–885 (1997). tetragonal space group P4 2 2, with unit cell dimensions: a = b 3 1 14. Okutani, T., Nagai, H., Mamiya, M., Shibuya, M. & Castillo, M. Effect of microgravity 75.97 Å, c = 34.87 Å, α = β = γ = 90°, and contained one polypeptide and magnetic field on the metallic and crystalline structure of magnetostrictive chain per asymmetry unit. Earth-grown crystals of unit cell dimensions: a SmFe2 synthesized by unidirectional solidification. Ann. N. Y. Acad. Sci. 1077, = b 78.86 Å, c = 37.15 Å, α = β = γ = 90°, belonged to the same space 146–160 (2006). group but showed comparatively better X-ray diffraction power to a 15. Ahari, H. et al. Effect of microgravity on the crystallization of a self-assembling minimum Bragg spacing of 1.6 Å. The structure of the 1 g lysozyme was layered material. Nature 388, 857–860 (1997). solved by the molecular replacement method with EPMR using the 16. Holcomb, J. et al. Protein crystallization: eluding the bottleneck of X-ray crystal- polypeptide chain of PDB: 5KXK as the search model. Residues 1–129 lography. AIMS Biophys. 4, 557–575 (2017). were unambiguously traced in the electron density map of the 1 g 17. Snell, E. H. & Helliwell, J. R. Macromolecular crystallization in microgravity. Rep. lysozyme, which was then used to phase the μg crystal data with Prog. Phys. 68, 799–853 (2005). Phaser . The latter could be traced for the polypeptide length except 18. McPherson, A. & DeLucas, L. J. 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Crystallogr. 26, 283–291 (1993). s41526-020-0102-3. 41. Delano, W. Pymol: an open-source molecular graphics tool. CCP4 Newsl. Protein Crystallogr. 40,82–92 (2002). Correspondence and requests for materials should be addressed to D.F. 42. Burley, S. K. et al. RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, bio- Reprints and permission information is available at http://www.nature.com/ technology and energy. Nucleic Acids Res. D1, D464–D474 (2019). reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims ACKNOWLEDGEMENTS in published maps and institutional affiliations. This study was funded by the Stanford Student Space Initiative (SSI) organization. We thank H. Perry at the Stanford Environmental Health & Safety Surplus Chemical Program for some of the reagents used in the study. We apologize to all those investigators whose papers could not be quoted in this article due to space limitations. We thank two anonymous reviewers for their constructive comments. Use Open Access This article is licensed under a Creative Commons of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Attribution 4.0 International License, which permits use, sharing, Laboratory, is supported by the US Department of Energy, Office of Science, Office of adaptation, distribution and reproduction in any medium or format, as long as you give Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural appropriate credit to the original author(s) and the source, provide a link to the Creative Molecular Biology Program is supported by the DOE Office of Biological and Commons license, and indicate if changes were made. The images or other third party Environmental Research, and by the National Institutes of Health, National Institute of material in this article are included in the article’s Creative Commons license, unless General Medical Sciences (including P41GM103393). The contents of this publication indicated otherwise in a credit line to the material. If material is not included in the are solely the responsibility of the authors and do not necessarily represent the article’s Creative Commons license and your intended use is not permitted by statutory official views of NIGMS or NIH. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. AUTHOR CONTRIBUTIONS A.L., J.M., and D.F. designed the study. A.L., J.M., S.R., K.H., and D.F. carried out © The Author(s) 2020 experiments. D.F. contributed reagents/materials/analysis tools. D.F. wrote the paper. 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