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In 2005 we reviewed microgravity for macromolecular crystallization, four years after the final flight of the Space Shuttle Orbiter, and five years before the first commercial flight to the Interna...

The Machine Recognition of Crystallization Outcomes (MARCO) initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups. Here, state-of-the-art machine learning algorithms are trained and tested on different parts of this data set. We find that more than 94% of the test images can be correctly labeled, irrespective of their experimental origin. Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments, this approach opens the door to both industrial and fundamental research applications., 11 pages, 4 figures, minor text and figure updates

As technology advances, the crystal volume that can be used to collect useful X-ray diffraction data decreases. The technologies available to detect and study growing crystals beyond the optical resolution limit and methods to successfully place the crystal into the X-ray beam are discussed., Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain ofSaccharomyces cerevisiaeglutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

Development of an ontology for the description of crystallization experiments and results is proposed., When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world’s crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.

Mapping crystallization results in chemical space helps to correlate seemingly distant relationships between crystallization conditions, points to possible optimization strategies and reveals promising unsampled areas of crystallization space., Macromolecular crystallization screening is an empirical process. It often begins by setting up experiments with a number of chemically diverse cocktails designed to sample chemical space known to promote crystallization. Where a potential crystal is seen a refined screen is set up, optimizing around that condition. By using an incomplete factorial sampling of chemical space to formulate the cocktails and presenting the results graphically, it is possible to readily identify trends relevant to crystallization, coarsely sample the phase diagram and help guide the optimization process. In this paper, chemical space mapping is applied to both single macromolecules and to a diverse set of macromolecules in order to illustrate how visual information is more readily understood and assimilated than the same information presented textually.

As part of a training set for automated image analysis, crystallization screening experiments for 269 different macromolecules were visually analyzed and a set of crystal images extracted. Outcomes and trends are analyzed., In the automated image analysis of crystallization experiments, representative examples of outcomes can be obtained rapidly. However, while the outcomes appear to be diverse, the number of crystalline outcomes can be small. To complement a training set from the visual observation of 147 456 crystallization outcomes, a set of crystal images was produced from 106 and 163 macromolecules under study for the North East Structural Genomics Consortium (NESG) and Structural Genomics of Pathogenic Protozoa (SGPP) groups, respectively. These crystal images have been combined with the initial training set. A description of the crystal-enriched data set and a preliminary analysis of outcomes from the data are described.

Spacecraft orbiting the earth experience a reduced acceleration environment due to being in a state of continuous free-fall. This state colloquially termed microgravity, has produced improved X-ray diffraction quality crystals of biological macromolecules. Improvements in X-ray diffraction resolution (detail) or signal to noise, provide greater detail in the three-dimensional molecular structure providing information about the molecule, how it works, how to improve its function or how to impede it. Greater molecular detail obtained by crystallization in microgravity, has important implications for structural biology. In this article we examine the theories behind macromolecule crystal quality improvement in microgravity using results obtained from studies with the model protein, chicken egg white lysozyme.

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.

Since the 1980s hundreds of macromolecular crystal growth experiments have been performed in the reduced acceleration environment of an orbiting spacecraft. Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown. Similarly, many samples have shown no improvement or degradation in comparison to those grown on the ground. A complex series of interrelated factors affect these experiments and by building a comprehensive archive of the results it was aimed to identify factors that result in success and those that result in failure. Specifically, it was found that dedicated microgravity missions increase the chance of success when compared with those where crystallization took place as a parasitic aspect of the mission. It was also found that the chance of success could not be predicted based on any discernible property of the macromolecule available to us.

The key concepts that attracted crystal growers, macromolecular or solid state, to microgravity research is that density difference fluid flows and sedimentation of the growing crystals are greatly reduced. Thus, defects and flaws in the crystals can be reduced, even eliminated, and crystal volume can be increased. Macromolecular crystallography differs from the field of crystalline semiconductors. For the latter, crystals are harnessed for their electrical behaviors. A crystal of a biological macromolecule is used instead for diffraction experiments (X-ray or neutron) to determine the three-dimensional structure of the macromolecule. The better the internal order of the crystal of a biological macromolecule then the more molecular structure detail that can be extracted. This structural information that enables an understanding of how the molecule functions. This knowledge is changing the biological and chemical sciences with major potential in understanding disease pathologies. Macromolecular structural crystallography in general is a remarkable field where physics, biology, chemistry, and mathematics meet to enable insight to the basic fundamentals of life. In this review, we examine the use of microgravity as an environment to grow macromolecular crystals. We describe the crystallization procedures used on the ground, how the resulting crystals are studied and the knowledge obtained from those crystals. We address the features desired in an ordered crystal and the techniques used to evaluate those features in detail. We then introduce the microgravity environment, the techniques to access that environment, and the theory and evidence behind the use of microgravity for crystallization experiments. We describe how ground-based laboratory techniques have been adapted to microgravity flights and look at some of the methods used to analyze the resulting data. Several case studies illustrate the physical crystal quality improvements and the macromolecular structural advances. Finally, limitations and alternatives to microgravity and future directions for this research are covered.

Reflection profiles were analyzed from microgravity-grown ( micro g) and earth-grown insulin crystals to measure mosaicity (eta) and to reveal mosaic domain structure and composition. The effects of cryocooling on single-domain and multi-domain crystals were compared. The effects of cryocooling on insulin structure were also re-examined. Microgravity crystals were of larger volume, were more homogeneous and were of higher quality than earth crystals. Several micro g crystals contained a single mosaic domain which encompassed all or nearly all of the crystal with an eta(avg) of 0.005 degrees. The earth crystals varied in quality and all contained multiple domains with an eta(avg) of 0.031 degrees. Cryocooling caused a 43-fold increase in eta for micro g crystals (eta(avg) = 0.217 degrees ) and an eightfold increase for earth crystals (eta(avg) = 0.246 degrees ). These results indicate that very well ordered crystals are not completely protected from the stresses associated with cryocooling, especially when structural perturbations occur. However, there were differences in the reflection profiles. For multi-mosaic domain crystals, each domain individually broadened and separated from the other domains upon cryocooling. Cryocooling did not cause an increase in the number of domains. A crystal composed of a single domain retained this domain structure and the reflection profiles simply broadened. Therefore, an improved signal-to-noise ratio for each reflection was measured from cryocooled single-domain crystals relative to cryocooled multi-domain crystals. The improved signal from micro g crystals, along with the increase in crystal size, facilitated the measurement of the weaker high-resolution reflections. The observed broadening of reflection profiles indicates increased variation in unit-cell parameters, which may be linked to cryocooling-associated structural changes and disorder.

Crystallization is a key step in macromolecular structure determination by crystallography. While a robust theoretical treatment of the process is available, due to the complexity of the system, the experimental process is still largely one of trial and error. In this article, efforts in the field are discussed together with a theoretical underpinning using a solubility phase diagram. Prior knowledge has been used to develop tools that computationally predict the crystallization outcome and define mutational approaches that enhance the likelihood of crystallization. For the most part these tools are based on binary outcomes (crystal or no crystal), and the full information contained in an assembly of crystallization screening experiments is lost. The potential of this additional information is illustrated by examples where new biological knowledge can be obtained and where a target can be sub-categorized to predict which class of reagents provides the crystallization driving force. Computational analysis of crystallization requires complete and correctly formatted data. While massive crystallization screening efforts are under way, the data available from many of these studies are sparse. The potential for this data and the steps needed to realize this potential are discussed., Comment: 9 pages, 3 figures

As preparation for an extensive study that aims to image the cryocooling process of macromolecular crystals, the ability to thermally image solid objects and liquids at temperatures far below 273 K is demonstrated. In the case of a large lysozyme crystal (1.0 x 0.7 x 0.2 mm), qualitative measurements show the cooling process to take about 0.6 s with the cooling taking place in a wave starting from the face of the crystal nearest to the origin of the cryostream and ending at the point furthest away from the origin. Annealing of this lysozyme crystal, cooled under good cryoprotectant conditions, shows that cold striations form perpendicular to the cooling stream. These striations become more pronounced after successive annealing. Cryocooling of a non-cryoprotected crystal of glucose isomerase displayed an 'S-shaped' cold front wave traveling across the sample. These preliminary results are qualitative but show the power of infrared imaging as a new tool for fundamental and practical cryocrystallography studies.

An X-ray based method for determining initial cryoprotectant concentrations necessary to protect solutions from crystalline ice formation on flash cooling was developed. X-ray images from a charge-coupled device (CCD) area detector were integrated as powder patterns and quantified by determining the standard deviation of the slope of the normalized intensity curve in the resolution range where ice rings are known to occur. The method was tested by determining the concentrations of glycerol, PEG400, ethylene glycol and 1,2-propanediol necessary to form an amorphous glass at 100 K with each of the 98 crystallization solutions of Crystal Screens I and II (Hampton Research, Laguna Hills, California, USA). For conditions that required glycerol concentrations of 35% or above, cryoprotectant conditions using (2R,3R)-(−)-2,3-butanediol were determined. The method proved to be remarkably reliable. The results build on previous work [Garman & Mitchell (1996).J. Appl. Cryst.29, 584–587] and extend the number of suitable starting conditions to alternative cryoprotectants.

This paper reports results from the first biological crystal-growth experiment on the International Space Station (ISS). Crystals of thaumatin were grown using liquid–liquid diffusion in Tygon® tubing transported in the Enhanced Gaseous Nitrogen Dewar (EGN). Different volume ratios and concentrations of protein and precipitant were used to test different adaptations of the vapor-diffusion crystallization recipe to the liquid–liquid diffusion method. The EGN warmed up from 77 to 273 K in about 4 d, about the same time it took to warm from 273 to 293 K. The temperature within the EGN was 293–297 K for the majority of the experiment. Air gaps that blocked liquid–liquid diffusion formed in the tubes. Nonetheless, crystals were grown. Synchrotron diffraction data collected from the best space-grown crystal extended to 1.28 A, comparable to previous studies of space-grown thaumatin crystals. The resolution of the best ground-control crystal was only 1.47 A. It is not clear if the difference in diffraction limit arises from factors other than crystal size. Improvements in temperature control and the elimination of air gaps are needed, but the results show that the EGN on the ISS can be used to produce space-grown crystals that diffract to high resolution.

The rich history of crystallization and how that history influences current practices is described. The tremendous impact of crystallization screens on the field is discussed., While crystallization historically predates crystallography, it is a critical step for the crystallographic process. The rich history of crystallization and how that history influences current practices is described. The tremendous impact of crystallization screens on the field is discussed.

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be time-consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and to the insertion and correct placement of sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient, CDcoeff, appears to be robust in this application, and coupled with hierarchical clustering and the overlay of crystallization outcome, reveals information of biological relevance. While tested with a single example the potential applications related to crystallography appear promising and the distance coefficient, clustering, and hierarchal visualization of results undoubtedly have applications in wider fields.

Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Four different crystal morphologies have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed, Crystals grown at 15 C were generally tetragonal, with space group P43212. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P21212 1. The tetragonal much less than orthorhombic morphology transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 0.8 -1.2M magnesium sulfate at pH 7.6 - 8.0 gave a hexagonal (trigonal) crystal form, space group P3121, which diffracted to 2.8 A. Ammonium sulfate was also found to result in a monoclinic form, space group C2. Small twinned monoclinic crystals of approx. 0.2 mm on edge were grown by dialysis followed by seeded sitting drop crystallization.

Protein biological function depends on structural flexibility and change. From cellular communication through membrane ion channels to oxygen uptake and delivery by haemoglobin, structural changes are critical. It has been suggested that vibrations that extend through the protein play a crucial role in controlling these structural changes. While nature may utilize such long-range vibrations for optimization of biological processes, bench-top characterization of these extended structural motions for engineered biochemistry has been elusive. Here we show the first optical observation of long-range protein vibrational modes. This is achieved by orientation-sensitive terahertz near-field microscopy measurements of chicken egg white lysozyme single crystals. Underdamped modes are found to exist for frequencies10 cm(-1). The existence of these persisting motions indicates that damping and intermode coupling are weaker than previously assumed. The methodology developed permits protein engineering based on dynamical network optimization.

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.

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis., National Institutes of Health (U.S.) (Protein Structure Initiative, NIGMS grant U54 GM094597), National Institutes of Health (U.S.) (grant NIH R01GM088396), National Science Foundation (U.S.) (Grant NSF CHE-1062607), National Science Foundation (U.S.) (Grant No. NSF DMR-1055586)

Observations of crystallization experiments are classified as specific outcomes and integrated through a phase diagram to visualize solubility and thereby direct subsequent experiments. Specific examples are taken from our high-throughput crystallization laboratory which provided a broad scope of data from 20 million crystallization experiments on 12 500 different biological macromolecules. The methods and rationale are broadly and generally applicable in any crystallization laboratory. Through a combination of incomplete factorial sampling of crystallization cocktails, standard outcome classifications, visualization of outcomes as they relate chemically, and application of a simple phase diagram approach, we demonstrate how to logically design subsequent crystallization experiments.

A program,AutoSherlock, has been developed to present crystallization screening results in terms of chemical space. This facilitates identification of lead conditions, rational interpretation of results and directions for the optimization of crystallization conditions.

As part of a training set for automated image analysis, ∼150 000 images of crystallization experiments from 96 diverse macromolecules have been visually classified within seven categories. Outcomes and trends are analyzed., Structural crystallography aims to provide a three-dimensional representation of macromolecules. Many parts of the multistep process to produce the three-dimensional structural model have been automated, especially through various structural genomics projects. A key step is the production of crystals for diffraction. The target macromolecule is combined with a large and chemically diverse set of cocktails with some leading ideally, but infrequently, to crystallization. A variety of outcomes will be observed during these screening experiments that typically require human interpretation for classification. Human interpretation is neither scalable nor objective, highlighting the need to develop an automatic computer-based image classification. As a first step towards automated image classification, 147 456 images representing crystallization experiments from 96 different macromolecular samples were manually classified. Each image was classified by three experts into seven predefined categories or their combinations. The resulting data where all three observers are in agreement provides one component of a truth set for the development and rigorous testing of automated image-classification systems and provides information about the chemical cocktails used for crystallization. In this paper, the details of this study are presented.

Flash-cooling of macromolecular crystals often compromises diffraction quality by increasing the mosaicity. In some cases, cycling the crystal between low temperature (LT) and room temperature (RT) can reverse this increase in mosaicity. Previous studies of RT/LT cycling have focused on the quality of the crystal as it was repeatedly returned to the LT state. Here, crystal quality is explored not only at LT but also when the crystal is returned to RT. The domain model is used to extract information about crystal order from reflection profiles measured from crystals of Escherichia coli beta-galactosidase at both temperatures. Despite optimization of the cryocooling protocol, the mosaicity increases by about sixfold with cooling and is anisotropic at both temperatures. The mosaicity increase is the consequence of a decrease in domain volume, an increase in the variation of domain cell dimensions and an increase in the angular spread between domains. Upon rewarming, the mosaicity recovers substantially, including the somewhat surprising recovery of domain volume, but incompletely. Over multiple RT/LT cycles disorder in both states increases, which appears to mainly arise from radiation damage, although a contribution from cool-thaw processes cannot be ruled out. The analysis further suggests that LT disorder is governed by variability inherent in the cooling process combined with the overall history of the crystal. In contrast, RT disorder appears to be governed principally by the overall history of the crystal. This suggests that with these particular crystals under the experimental conditions used, particularly at high-intensity synchrotron X-ray sources, RT/LT cycling annealing protocols should involve few cycles so as to limit the hysteresis in both temperature states while taking advantage of the inherent variability in the cooling process that may result in improved crystal order at LT.

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.

Lysozyme has been used to investigate the effect of microgravity crystallisation on protein crystal perfection. Crystals were grown in the European Space Agency's Advanced Protein Crystallisation Facility onboard the NASA Space Shuttle. Two missions of differing duration took place, Spacehab-1 and IML-2. The microgravity crystallisation time in each was 7 days and 10 hours, and 12 days and 11 hours respectively. The IML-2 crystals had grown much larger than the Spacehab-1 crystals (2.5 mm versus 0.8 mm at maximum). The earth grown control crystals, in each case, reached a size of 0.8 mm at maximum. The perfection of the crystals was evaluated with collimated, intense, synchrotron radiation. This was done using the Laue method, via the spot size, and by monochromatic rocking widths directly. For the Spacehab-1 crystals spot size measurements were carried out on station 9.5 of the SRS, along with an analysis of intensity to sigma ratio, immediately after the mission. Five months later rocking widths were measured at LURE. The IML-2 crystals were evaluated at the ESRF, on BL3 three months after their return to earth and also a further three months later on the joint Swiss-Norwegian beamline. Both the Spacehab-1 and IML-2 crystals were of exceptional perfection with the crystal mosaicity reaching values as small as 0.0010° and 0.0017° respectively. Earth-grown control crystals had values as small as 0.0032° and 0.007° respectively. There is no evidence of ‘shelf-life’ ageing of the crystals, at least over a period of 6 months, since there is close agreement of the mosaicity values from the Spacehab-1 crystals tested within weeks of that mission and the IML-2 crystals tested 6 months after that mission. The perfect mosaic block size has evidently increased over that realised in the earth-grown controls.

Neutron diffraction is uniquely sensitive to hydrogen positions and protonation state. In that context structural information from neutron data is complementary to that provided through X-ray diffraction. However, there are practical obstacles to overcome in fully exploiting the potential of neutron diffraction, i.e. low flux and weak scattering. Several approaches are available to overcome these obstacles and we have investigated the simplest: increasing the diffracting volume of the crystals. Volume is a quantifiable metric that is well suited for experimental design and optimization techniques. By using response surface methods we have optimized the xylose isomerase crystal volume, enabling neutron diffraction while we determined the crystallization parameters with a minimum of experiments. Our results suggest a systematic means of enabling neutron diffraction studies for a larger number of samples that require information on hydrogen position and/or protonation state.

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine φ slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-­fold higher reflection peak heights and diffracted to significantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

A challenge in neutron diffraction experiments is growing large (greater than 1 cu mm) macromolecule crystals. In taking up this challenge we have used statistical experiment design techniques to quickly identify crystallization conditions under which the largest crystals grow. These techniques provide the maximum information for minimal experimental effort, allowing optimal screening of crystallization variables in a simple experimental matrix, using the minimum amount of sample. Analysis of the results quickly tells the investigator what conditions are the most important for the crystallization. These can then be used to maximize the crystallization results in terms of reducing crystal numbers and providing large crystals of suitable habit. We have used these techniques to grow large crystals of Glucose isomerase. Glucose isomerase is an industrial enzyme used extensively in the food industry for the conversion of glucose to fructose. The aim of this study is the elucidation of the enzymatic mechanism at the molecular level. The accurate determination of hydrogen positions, which is critical for this, is a requirement that neutron diffraction is uniquely suited for. Preliminary neutron diffraction experiments with these crystals conducted at the Institute Laue-Langevin (Grenoble, France) reveal diffraction to beyond 2.5 angstrom. Macromolecular crystal growth is a process involving many parameters, and statistical experimental design is naturally suited to this field. These techniques are sample independent and provide an experimental strategy to maximize crystal volume and habit for neutron diffraction studies.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0–5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105proteins that is underway, raises expectations for equivalent three dimensional structural databases.

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.