Advanced Protein Crystallization Facility Research Articles

Phase behavior and crystallogenesis under counter-diffusion conditions of the collagen-model peptide (Pro–Pro–Gly) 10

Single chains of the collagen-model polypeptide with sequence (Pro–Pro–Gly) 10, hereafter referred to as (PPG) 10, aggregate to form rod-shaped triple helices. Precipitation diagrams of (PPG) 10 as a function of pH and various molecular mass poly(ethylene glycol) concentrations have been measured. These results were used to design experiments to test the quality of (PPG) 10 crystal grown on ground (in gelled and free solutions) and in space in two different facilities, the Advanced Protein Crystallization Facility based on the free-interface diffusion technique and the Granada Crystallization Facility based on counter-diffusion technique. Experiments were monitored by optical microscopy on earth. X-ray diffraction analysis of the grown crystals reveals the benefit of the counter-diffusion technique.

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 3. Analysis of residual acceleration-induced motion.

(PPG)(10) crystallization experiments onboard the ISS using the Advanced Protein Crystallization Facility have shown parallel and coherent crystal motions. The residual acceleration profiles and the History of the ISS Increment 3 mission allow a quantitative interpretation of these motions. Two events determine the observed crystal motions: the undocking of the Space Shuttle and a change in the ISS attitude required for power generation. No correlation between these motions and the crystal quality is apparent.

Video observation of protein crystal growth in the advanced protein crystallization facility aboard the space shuttle mission STS-95

Crystals of polypeptide with sequence (Pro-Pro-Gly) 10 were grown in the Advanced Protein Crystallization Facility during the US Space Shuttle STS-95 Mission. Well diffracting crystals were obtained by dialysis; they provided the highest resolution X-ray data so far collected for a collagen-like triple helical structure (Berisio et al., Acta Crystallogr. D 56 (2000) 55). During the Mission the crystal growth was monitored by a CCD video camera. The image analysis provided information on spatial distribution of crystals, their movements and their growth. The spatial distribution was non-uniform inside the reactors. Limited motions were observed. The linear growth rate indicates that, in some cases, the growth did not cease in the microgravity environment.

Structure of tetragonal hen egg-white lysozyme at 0.94 A from crystals grown by the counter-diffusion method.

Very high quality crystals of tetragonal hen egg-white lysozyme were grown in the Advanced Protein Crystallization Facility (APCF) on board the Space Shuttle using a modified free-interface diffusion (FID) reactor designed ad hoc to have a longer diffusion path. This design allows the performance of true counter-diffusion experiments. Crystals were obtained under the classical chemical conditions defined 50 y ago with NaCl as a crystallizing agent and acetate pH 4.5 as a buffer. Counter-diffusion crystallization allows a "physical" instead of chemical optimization of growth conditions: indeed, this method screens for the best supersaturation conditions in a single trial and yields crystals of very high quality. A complete diffraction data set was collected at atomic resolution from one of these crystals using synchrotron radiation at the DESY-EMBL beamlines. The overall R(merge) on intensities in the resolution range 31-0.94 A was 5.2% and the data were 98.9% complete. Refinement was carried out with the programs CNS and SHELX97 to a final crystallographic R factor of 12.26% for 72 390 reflections. A mean standard uncertainty in the atomic positions of 0.024 A was estimated from inversion of blocked least-squares matrices. 22 side chains show alternate conformations and the loop 59-75 adopts in the same crystal packing two conformations that were observed for either triclinic or tetragonal lysozyme in previous high-resolution studies. In addition to 255 water molecules, the crystallizing agent (one hexacoordinated sodium ion and five chloride anions) participates in the ordered lysozyme hydration shell.

Nucleation and growth of thaumatin crystals within a gel under microgravity on STS-95 mission vs. under Earth's gravity

In the frame of a study of the effects of microgravity on protein crystallization, tetragonal thaumatin crystals were, for the first time, prepared in agarose gel within the Advanced Protein Crystallization facility (APCF) on Space Shuttle mission STS-95 (October 1998). We have reported elsewhere that these crystals are of superior crystallographic quality, with regard to their diffraction intensity and limit as well as mosaic spread, when compared to control crystals grown on Earth [Lorber et al ., Acta Crystallogr. (1999) D55, 1491]. The analysis of images taken during the growth of these crystals under microgravity indicates that their nucleation was more synchronous than that of control crystals which were prepared simultaneously on Earth under otherwise identical conditions. Nucleation occurred essentially in the bulk of the medium and crystal growth proceeded up to 2 times faster. Crystals prepared under microgravity had better optical properties than control crystals. Experimental results give evidence of the existence of zones depleted in protein located around growing crystals. Also, they are in favor of a correlation between the better diffraction properties of these crystals and the more favorable crystallization parameters in Space. The content of individual crystals was analyzed and the results of purity analyses are presented. The advantages of crystallization in a gel under microgravity and importance of image documentation for asserting growth cessation are discussed.

Effects of microgravity on the crystal quality of a collagen-like polypeptide.

(Pro-Pro-Gly)(10) is one of the most widely studied collagen polypeptide models. Microgravity crystal growth of (Pro-Pro-Gly)(10) was carried out in the Advanced Protein Crystallization Facility aboard the Space Shuttle Discovery during the STS-95 mission. Crystals were successfully grown in all experiments, using both dialysis and free-interface diffusion methods. The quality of the microgravity-grown crystals and of ground-grown counterparts was assessed by X-ray synchrotron diffraction. Microgravity-grown crystals exhibited a significant improvement in terms of dimensions and resolution limit. As previously reported, crystals were orthorhombic, space group P2(1)2(1)2(1). However, the diffraction pattern showed weak reflections, never previously measured, that were consistent with new unit-cell parameters a = 26.9, b = 26.4, c = 182.5 A. This allowed the derivation of a new model for the arrangement of the triple-helical molecules in the crystals.

Growth kinetics and motion of thaumatin crystals during USML-2 and LMS microgravity missions and comparison with earth controls

As part of a study of the effects of microgravity on protein crystallization, the growth of tetragonal crystals of thaumatin was monitored by CCD-time-lapse video in environments where convection is negligible. In Space Shuttle missions entitled United States Microgravity Laboratory-2 and Life and Microgravity Sciences, free interface diffusion and dialysis techniques were utilized to grow crystals in the advanced protein crystallization facility (APCF). Ng et al. (Acta Crystallogr. D 53 (1997) 724) have shown that the crystals recovered in these experiments are of superior crystallographic quality (at the level of their diffraction intensity, resolution, and mosaicity) with regard to earth controls. Here, the number of crystals, their size, growth rate, and protein solubility in microgravity were compared with data of dialysis experiments performed in parallel on earth. Image analysis shows that in microgravity about one-quarter to half of the crystals have nucleated and grown in the bulk of the solution, the remaining being attached to the walls of crystallization vessels. The growth of free-floating crystals was 2.5 times faster, has resulted in 15-fold larger crystals, and consumed more protein than that of attached crystals in earth controls. Distances between immobile free-floating crystals in microgravity were related to the size of the latter. Experimental results are in favor of a correlation between more favorable growth parameters in microgravity and better diffraction properties. The displacements of free-floating crystals at various velocities and in various directions on unrelated trajectories are indicative of drift and stirring motion. On the basis of an overview of reactors monitored on four APCF missions some forces causing motion are proposed. Advantages of crystallization in microgravity are discussed and recommendations for future experiments given.

Crystallization within agarose gel in microgravity improves the quality of thaumatin crystals.

To prevent crystals from moving in orbit and sedimenting upon their return to earth, the model protein thaumatin was crystallized in agarose gel in the Advanced Protein Crystallization Facility during the eight-day Space Shuttle mission STS-95 (November 1998). The quality of tetragonal crystals grown in microgravity was compared with that of controls prepared in parallel in the laboratory. On the basis of their diffraction properties, microgravity crystals were more ordered than crystals grown in gel on earth (the latter being, on average, better than reference crystals obtained in solution on earth). It is concluded that protein crystallization within a gel in microgravity may yield crystals of superior quality by combining the advantages of both environments. A possible explanation for the positive effect of microgravity on protein crystallization in gels involving the better quality of the nucleus is discussed.

Crystal growth studies in microgravity with the APCF. II. Image analysis studies

Lysozyme crystallization experiments performed in the Advanced Protein Crystallization Facility during SpaceHab-01 mission are analyzed using pictures obtained during the flight. The temporal evolution of experiments performed using free interface diffusion (FID) and dialysis (DIA) techniques was studied to gain a deeper insight into the processes of nucleation and growth. Heterogeneous nucleation on the walls of the reactor predominates over nucleation in the bulk solution. Nucleation takes place simultaneously throughout the protein chamber, which means that the supersaturation inside the reactors has reached homogeneity before nucleation. In terms of precipitation behavior this means that the APCF reactors are working as very slow-mixing batch experiments. Measured growth rates range from 10 to 50Å/s and decrease asymptotically to zero. L (a linear dimension of the crystals) versus time1/2 plots are discussed. Large-scale growth rate fluctuations are observed and explained by the interaction between diffusive fields of neighboring crystals. Crystals not attached to the reactor walls are observed to move, with varying velocities and directions of movement, and at a given time, undergo a fast acceleration that projects them towards the walls of the reactor. The amount of information that can be derived from the final data set supplied after the space experiment is limited by the technical features implemented in the APCF. Some recommendations to enhance these features are proposed.

Crystal growth studies in microgravity with the APCF. I. Computer simulation of transport dynamics

A computer program has been designed to simulate the pre-nucleation history of protein crystal growth experiments in the Advanced Protein Crystallization Facility developed by ESA to conduct crystallization experiments under microgravity conditions. The program was fed with the initial conditions of a set of experiments performed by the ESA Diagnostic Group during the SpaceHab-01 mission. The time histories of the salt and protein concentrations and supersaturation at any point inside the reactor were obtained. The differences between Dialysis and Free Interface Diffusion techniques are discussed. Both DIA and FID expriments performed in the short protein chamber of the APCF are basically very slow mixing batch experiments although differences exist in the equilibration behavior. The kinetics of protein nucleation coupled with transport phenomena are analyzed, showing that the supersaturation rate modifies the supersaturation value at which nucleation occurs as well as waiting times for nucleation. Reactor geometry and initial conditions can be optimized. As a general rule, higher protein concentrations will produce crystals that nucleate at lower supersaturation and supersaturation rates, provided that the growth chamber is long enough to implement a true diffusive set-up. When these conditions are fulfilled, the growth system becomes self regulated and crystals nucleate under different supersaturation and supersaturation rate conditions, performing a kind of automatic, single-reactor screening experiment.

CCD video observation of microgravity crystallization of lysozyme and correlation with accelerometer data.

Lysozyme has been crystallized using the ESA Advanced Protein Crystallization Facility onboard the NASA Space Shuttle Orbiter during the IML-2 mission. CCD video monitoring was used to follow the crystallization process and evaluate the growth rate. During the mission some tetragonal crystals were observed moving over distances of up to 200 micrometers. This was correlated with microgravity disturbances caused by firings of vernier jets on the Orbiter. Growth-rate measurement of a stationary crystal (which had nucleated on the growth reactor wall) showed spurts and lulls correlated with an onboard activity: astronaut exercise. The stepped growth rates may be responsible for the residual mosaic block structure seen in crystal mosaicity and topography measurements.

Crystallogenesis studies in microgravity with the Advanced Protein Crystallization Facility on SpaceHab-01

The Advanced Protein Crystallization Facility (APCF), a new protein crystallization device developed by ESA for the IML-2 Mission in 1994, was tested in its maiden flight on STS-57 Mission in SpaceHab-01 with a physico-chemical experiment on lysozyme crystallization. In pre-flight ground experiments, prior to the Shuttle Mission, the protocol for lysozyme crystallization with NaCl was based on its solubility diagram at 18°C and pH 4.5. Crystallization was conducted under microgravity in 25 APCF reactors using vapor diffusion, dialysis, and free liquid interface diffusion, with control on earth in 25 identical reactors. Identical supersaturation values were tested by the three crystallization techniques. Values of supersaturation derived from ground experiments allowed for conditions that yielded crystals in microgravity. The average number and size of crystals from the flight experiment and the earth control showed no significant difference; however many crystals were not free floating and grew on the walls of some of the protein chambers. The dialysis technique proved to be suitable, since no additional nucleation was generated by the membrane. Protein concentration measurements indicated that 13 days after activation of the experiment as much as 70–90% of the protein in supersaturated state had already crystallized. Data indicated differences in the crystallization behavior depending upon the crystallization set-up. Images of the protein chamber of 6 reactors, recorded during the flight, allowed us to evaluate the early stage of crystallization, to verify that recovered crystals had actually grown under microgravity conditions, and showed motions of crystals during the Mission. Using synchrotron radiation, resolution and rocking curve measurements of ground and space lysozyme crystals grown in APCF reactors showed no significant differences, although the values are much better than previously recorded diffraction limits and mosaicity data obtained with tetragonal lysozyme crystals grown in other set-ups and under different conditions. All controls foreseen throughout the microgravity experiment proved to be essential for the interpretation of the flight data, as concerning the effect of microgravity.

Crystallographic analyses of lysozyme and collagenase microgravity grown crystals versus ground controls

Hen egg white lysozyme and collagenase from Hypoderma lineatum larvae have been used as model proteins of known physical and chemical properties to study the influence of microgravity on crystal quality. This was performed using the Advanced Protein Crystallization Facility instrument during two successive missions of the US Space Shuttle. Lysozyme crystals were grown during the SpaceHab-01 mission and are compared with those obtained during the six months Eureca mission using the Protein Crystallization Facility instrument. Collagenase crystals were produced during the IML-2 mission. Space grown crystals as well as grounds controls were analyzed using synchrotron radiation to assess their diffraction limits. For lysozyme crystals, a large number of ground and microgravity grown crystals were analyzed and showed nearly equivalent maximum resolution (best one is 1.3 Å). Collagenase crystals from IML-2, although presenting a marginal increase of resolution (1.7 Å), display a better signal to noise ratio than ground crystals.

Visualization of protein crystal growth inside hanging-drop reactors of the Advanced Protein Crystallization Facility

The Advanced Protein Crystallization Facility is widely used by European and US teams to study protein crystal growth under microgravity conditions. This paper describes the laboratory equipment developed to observe the crystals, by means of a charge-coupled-device camera, during the growth process on the ground inside the hanging-drop reactors of the Advanced Protein Crystallization Facility. The apparatus was used during ground tests performed to prepare the microgravity experimentation, allowing a time reduction in determining the crystallization conditions to be used for the microgravity experiment.

CCD video observation of microgravity crystallization: apocrustacyanin C 1

Apocrustacyanin C 1 has been crystallized in the vapour-diffusion apparatus of ESA's Advanced Protein Crystallization Facility (APCF) on-board the NASA space shuttle STS-65 International Microgravity Laboratory-2 (IML-2) mission. Crystal growth was monitored by black and white CCD observation at time intervals throughout the experiment. The resulting crystals displayed a motion within the hanging drop that is attributed to Marangoni convection effects. The images also show a “halo” effect around the growing crystals which can be attributed to the presence of depletion zones i.e. solution regions which are depleted of this coloured protein.

High-resolution structure (1.33 A) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission.

Crystals of tetragonal hen egg-white lysozyme were grown using Advanced Protein Crystallization Facility (APCF) apparatus under a microgravity environment (SpaceHab-01 mission) and ground control conditions. Crystals were grown from NaCl as a crystallizing agent at pH 4.3. The X-ray diffraction patterns of the best diffracting ground- and space-grown crystals were recorded using synchrotron radiation and an image plate on the W32 beamline at LURE. Both ground- and space-grown crystals showed nearly equivalent maximum resolution of 1.3-1.4 A. Refinements were carried out with the program X-PLOR with final R values of 18.45 and 18.27% for structures from ground- and space- grown crystals, respectively. The two structures are nearly identical with the root-mean-square difference on all protein atoms being 0.13 A. Some residues of the two refined structures show multiple alternative conformations. Two ions were localized into the electron-density maps of the two structures: one chloride ion at the interface between two symmetry-related molecules and one sodium ion stabilizing the loop Ser60-Leu75. The sodium ion is surrounded by six ligands which form a bipyramid around it at distances of 2.2-2.6 A.

Lysozyme crystal growth kinetics monitored using a Mach-Zehnder interferometer.

A Mach-Zehnder interferometer has been developed for the monitoring of the kinetics of the diffusion process in protein crystal growth. This device can be used in conjunction with the ESA Advanced Protein Crystallization Facility (APCF), which allows experiments under microgravity conditions (e.g. on board the NASA Space Shuttle). Experimental trials on the ground have been carried out with the interferometer using the engineering model of the APCF and a protein dialysis reactor. Chicken egg-white lysozyme crystal growth, as a test, has thereby been monitored directly. The changes of concentration in the solution over time have been determined via the refractive index measurements made and subsequently correlated with visual monitoring of crystal growth in a repeat experiment.

Crystallization of apocrustacyanin on the International Microgravity Laboratory (IML-2) mission.

Rod-shaped crystals of apocrustacyanin C1 have been grown under microgravity on the International Microgravity Laboratory (IML-2) NASA space shuttle mission using the vapour-diffusion set-up of the Advanced Protein Crystallization Facility (APCF). The crystals obtained under microgravity are compared with crystals grown simultaneously in ground control experiments in identical APCF reactors, and with those obtained in the laboratory. The degree of reproducibility of the results in microgravity was also tested. Statistically, the microgravity-grown crystals are larger and of better X-ray diffraction quality than those grown in the ground controls but inferior to the best crystals grown in sitting drops, in the laboratory. Diffracting crystals, the best to 2.3 A, were produced in seven out of the eight reactors in microgravity, whereas the eight ground control reactors yielded only one poorly formed crystal suitable for diffraction studies, which also diffracted to 2.3 A. The crystals belong to the space group P2(1)2(1)2(1) with two subunits per asymmetric unit.

Protein and virus crystal growth on international microgravity laboratory-2

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

Experiment equipment for protein crystallization in μ g facilities

For protein crystal growth under microgravity conditions, experiment equipment is needed which provides common techniques for crystallization, and simultaneously complies with the requirements for space flight experiments. For the Advanced Protein Crystallization Facility (APCF) to be flown in a middeck locker of the Space Shuttle (Spacehab and IML-2 mission), three types of protein reactors have been developed. These allow protein crystallization according to the methods liquid-liquid diffusion, dialysis, and vapour diffusion (hanging drop). Due to identical mechanical and optical interfaces, the experimenter may individually select type and size of the reactors for his experiments. Additionally, a new type of hanging drop reactor was designed which allows active control of the supersaturation inside the protein solution.