Efficient protein crystallization

High-throughput molecular biology and crystallography advances have placed an increasing demand on crystallization, the one remaining bottleneck in macromolecular crystallography. This paper describes three experimental approaches, an incomplete factorial crystallization screen, a high-throughput nanoliter crystallization system, and the use of a neural net to predict crystallization conditions via a small sample (approximately 0.1%) of screening results. The use of these technologies has the potential to reduce time and sample requirements. Initial experimental results indicate that the incomplete factorial design detects initial crystallization conditions not previously discovered using commercial screens. This may be due to the ability of the incomplete factorial screen to sample a broader portion of "crystallization space," using a multidimensional set of components, concentrations, and physical conditions. The incomplete factorial screen is complemented by a neural network program used to model crystallization. This capability is used to help predict new crystallization conditions. An automated, nanoliter crystallization system, with a throughput of up to 400 conditions/h in 40-nl droplets (total volume), accommodates microbatch or traditional "sitting-drop" vapor diffusion experiments. The goal of this research is to develop a fully-automated high-throughput crystallization system that integrates incomplete factorial screen and neural net capabilities.     

Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens

Recent efforts to collect and mine crystallization data from structural genomics (SG) consortia have led to the identification of minimal screens and novel screening strategies that can be used to streamline the crystallization process. Two groups, the Joint Center for Structural Genomics and the University of Toronto, carried out large-scale crystallization trials on different sets of bacterial targets (539, JCSG and 755, Toronto), using different sample processing and crystallization methods, and then analyzed their results to identify the smallest subset of conditions that would have crystallized the maximum number of protein targets. The JCSG Core Screen contains 67 conditions (from 480) while the Toronto Minimal Screen contains 6 (from 48). While the exact conditions included in the two screens do not overlap, the major precipitants of the conditions are similar and thus both screens can be used to determine if a protein has a natural propensity to crystallize. In addition, studies from other groups including the University of Queensland, the Mycobacterium tuberculosis SG group, the Southeast Collaboratory for SG, and the York Structural Biology Laboratory indicate that alternative crystallization strategies may be more successful at identifying initial crystallization conditions than typical sparse matrix screens. These minimal screens and alternative screening strategies are already being used to optimize the crystallization processes within large SG efforts. The differences between these results, however, demonstrate that additional studies which examine the influence of protein biophysical properties and sample preparation methods on crystal formation must also be carried out before more robust screens can be identified.     

Crystallization behavior of glucomannan

Five different glucomannan samples were recrystallized from dilute solution. Depending on the experimental conditions, the crystals obtained could be identified as corresponding to the mannan I (anhydrous precipitate of more or less regular lozenge-shaped crystals) or mannan II (hydrated gel-forming pseudo-fibrillar precipitate). High-molecular-weight material, low temperature of crystallization, or a polar crystallization medium favored the mannan II polymorph, whereas a low-molecular weight, a high temperature of crystallization, and a crystallization medium of low polarity yielded the mannan I polymorph. Since the base-plane unit-cell dimensions are fairly constant with respect to variation of glucose, it is likely that isomorphous replacement of mannose by glucose occurs in glucomannan crystallization; the data also indicate that perfection of the glucomannan crystals was reduced in specimens having a high glucose:mannose ratio. The oriented crystallization of glucomannan on cellulose microfibrils was also studied under conditions where the mannan I polymorph was obtained. This gave shish-kebab structures that were characterized.     

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.     

A model-based approach for mining membrane protein crystallization trials

MOTIVATION: Membrane proteins are known to play crucial roles in various cellular functions. Information about their function can be derived from their structure, but knowledge of these proteins is limited, as their structures are difficult to obtain. Crystallization has proved to be an essential step in the determination of macromolecular structure. Unfortunately, the bottleneck is that the crystallization process is quite complex and extremely sensitive to experimental conditions, the selection of which is largely a matter of trial and error. Even under the best conditions, it can take a large amount of time, from weeks to years, to obtain diffraction-quality crystals. Other issues include the time and cost involved in taking multiple trials and the presence of very few positive samples in a wide and largely undetermined parameter space. Therefore, any help in directing scientists' attention to the hot spots in the conceptual crystallization space would lead to increased efficiency in crystallization trials. RESULTS: This work is an application case study on mining membrane protein crystallization trials to predict novel conditions that have a high likelihood of leading to crystallization. We use suitable supervised learning algorithms to model the data-space and predict a novel set of crystallization conditions. Our preliminary wet laboratory results are very encouraging and we believe this work shows great promise. We conclude with a view of the crystallization space that is based on our results, which should prove useful for future studies in this area.     

Effects of solvent polarity on the absorption and fluorescence spectra of chlorogenic acid and caffeic acid compounds: determination of the dipole moments

The effects of solvent polarity on absorption and fluorescence spectra of biologically active compounds (chlorogenic acid (CGA) and caffeic acids (CA)) have been investigated. In both spectra pronounced solvatochromic effects were observed with shift of emission peaks larger than the corresponding UV‐vis electronic absorption spectra. From solvatochromic theory the ground and excited‐state dipole moments were determined experimentally and theoretically. The differences between the excited and ground state dipole moment determined by Bakhshiev, Kawski–Chamma–Viallet and Reichardt equations are quite similar. The ground and excited‐state dipole moments were determined by theoretical quantum chemical calculation using density function theory (DFT) method (Gaussian 09) and were also similar to the experimental results. The HOMO‐LUMO energy band gaps for CGA and CFA were calculated and found to be 4.1119 and 1.8732 eV respectively. The results also indicated the CGA molecule is more stable than that of CFA. It was also observed that in both compounds the excited state possesses a higher dipole moment than that of the ground state. This confirms that the excited state of the hydroxycinnamic compounds is more polarized than that of the ground state and therefore is more sensitive to the solvent. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein crystallization: virtual screening and optimization

Advances in genomics have yielded entire genetic sequences for a variety of prokaryotic and eukaryotic organisms. This accumulating information has escalated the demands for three-dimensional protein structure determinations. As a result, high-throughput structural genomics has become a major international research focus. This effort has already led to several significant improvements in X-ray crystallographic and nuclear magnetic resonance methodologies. Crystallography is currently the major contributor to three-dimensional protein structure information. However, the production of soluble, purified protein and diffraction-quality crystals are clearly the major roadblocks preventing the realization of high-throughput structure determination.

This paper discusses a novel approach that may improve the efficiency and success rate for protein crystallization. An automated nanodispensing system is used to rapidly prepare crystallization conditions using minimal sample. Proteins are subjected to an incomplete factorial screen (balanced parameter screen), thereby efficiently searching the entire “crystallization space” for suitable conditions. The screen conditions and scored experimental results are subsequently analyzed using a neural network algorithm to predict new conditions likely to yield improved crystals. Results based on a small number of proteins suggest that the combination of a balanced incomplete factorial screen and neural network analysis may provide an efficient method for producing diffraction-quality protein crystals.     

Shell wars II: the influence of relative size on decisions made during hermit crab shell fights

The value of contested resources (shells) in hermit crab fights depends on the sizes of the crabs relative to the sizes of the resources. Thus when relative contestant size is the main experimental variable, motivational factors associated with shell size will also be an experimental problem. Two experiments are described that together overcome this problem. Relative crab size influences all stages of shell fights including pre-fight display, escalation, eviction and examination of the opponent's shell by the victor both before and after eviction of the loser. Shell fights occur more often between disparately-sized animals than between those similar in size. This apparent contradiction of recent theory (Maynard-Smith & Parker 1976) is probably due to the high cost of being without a shell and the small chance that an escalated fight will result. Relative crab size influences the time taken in resource assessments and thus the effectiveness of these assessments is also probably influenced. Causal factors influencing each of the major decisions in shell fights are described and although these fights are more complex than most they are in general agreement with theory on animal contests.     

Simulations of a protein crystal: explicit treatment of crystallization conditions links theory and experiment in the streptavidin-biotin complex

A 250 ns molecular dynamics simulation of the biotin-liganded streptavidin crystal lattice, including cryoprotectant molecules and crystallization salts, is compared to a 250 ns simulation of the lattice solvated with pure water. The simulation using detailed crystallization conditions preserves the initial X-ray structure better than the simulation using pure water, even though the protein molecules display comparable mobility in either simulation. Atomic fluctuations computed from the simulation with crystallization conditions closely reproduce fluctuations derived from experimental temperature factors (correlation coefficient of 0.88, omitting two N-terminal residues with very high experimental B-factors). In contrast, fluctuations calculated from the simulation with pure water were less accurate, particularly for two of the streptavidin loops exposed to solvent in the crystal lattice. Finally, we obtain good agreement between the water and cryoprotectant densities obtained from the simulated crystallization conditions and the electron density due to solvent molecules in the X-ray structure. Our results suggest that detailed lattice simulations with realistic crystallization conditions can be used to assess potential function parameters, validate simulation protocols, and obtain valuable insights that solution-phase simulations do not easily provide. We anticipate that this will prove to be a powerful strategy for molecular dynamics simulations of biomolecules.     

Potential of different techniques of preferential crystallization for enantioseparation of racemic compound forming systems

Recently the feasibility of preferential crystallization for enantioseparation of racemic compound forming systems has been demonstrated (Lorenz et al., Application of preferential crystallization to resolve racemic compounds in a hybrid process. Chirality 2006;18:828-840; Polenske et al., Separation of the propranolol hydrochloride enantiomers by preferential crystallization: thermodynamic basis and experimental verification. Cryst Growth Des 2007;7:1628-1634). Here, the development and the potential of an efficient separation process operated via two different techniques of preferential crystallization are studied: (1) seeded isothermal preferential crystallization and (2) auto-seeded polythermal preferential crystallization. Both techniques were investigated in the batch and in the cyclic operation mode. On the example of mandelic acid as a typical racemic compound forming system, it is demonstrated that a cyclic auto-seeded polythermal process is feasible and significantly more efficient than the seeded isothermal one.     

An experimental test of the discreteness-of-charge effect in positive and negative lipid bilayers

The electrostatic properties of charged bilayers and the bilayer component of biological membranes are often described theoretically by assuming the charge is smeared uniformly over the surface. This is one of the fundamental assumptions in the Gouy-Chapman-Stern (GCS) theory. However, the average distance between the charged phospholipids in a typical biological membrane is 2-3 nm, which is 2-3 times the Debye length in a 0.1 M salt solution. Existing discreteness-of-charge theories predict significant deviations from the GCS theory for the adsorption of ions to such membranes. We considered the predictions of the simplest discreteness-of-charge theory [Nelson, A. P., & McQuarrie, D. A. (1975) J. Theor. Biol. 55, 13-27], in which the charges are assumed to be fixed in a square lattice and the potential is described by the linearized Poisson-Boltzmann relation. This theory predicts deviations that are larger for counterions than for co-ions and much larger for divalent than for monovalent counterions. We tested these predictions by measuring the adsorption of a fluorescent monovalent anion and a paramagnetic divalent cation to both positive and negative membranes, which we demonstrated experimentally had the same average surface potential. All our experimental results with probes, including those obtained on membranes in the gel rather than in the liquid-crystalline state, agreed with the predictions of the GCS theory rather than with the discreteness-of-charge theory. A simple calculation indicates that the agreement between the experimental results and the predictions of the GCS theory could be due to the finite size of the lipids.     

Unexpected differences between D- and L- tyrosine lead to chiral enhancement in racemic mixtures.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 degrees C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

Unexpected Differences between D- and L- Tyrosine Lead to Chiral Enhancement in Racemic Mixtures Dedicated to the memory of Prof. Shneior Lifson – A great liberal thinker.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 °C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

XtalPred: a web server for prediction of protein crystallizability

XtalPred is a web server for prediction of protein crystallizability. The prediction is made by comparing several features of the protein with distributions of these features in TargetDB and combining the results into an overall probability of crystallization. XtalPred provides: (1) a detailed comparison of the protein's features to the corresponding distribution from TargetDB; (2) a summary of protein features and predictions that indicate problems that are likely to be encountered during protein crystallization; (3) prediction of ligands; and (4) (optional) lists of close homologs from complete microbial genomes that are more likely to crystallize. AVAILABILITY: The XtalPred web server is freely available for academic users on http://ffas.burnham.org/XtalPred     

Microflow system promotes acetaminophen crystal nucleation

It is known that interfaces have various impacts on crystallization from a solution. Here, we describe crystallization of acetaminophen using a microflow channel, in which two liquids meet and form a liquid–liquid interface due to laminar flow, resulting in uniform mixing of solvents on the molecular scale. In the anti‐solvent method, the microflow mixing promoted the crystallization more than bulk mixing. Furthermore, increased flow rate encouraged crystal formation, and a metastable form appeared under a certain flow condition. This means that interface management by the microchannel could be a beneficial tool for crystallization and polymorph control.     

Propagation of Biochirality: Crossovers and Nonclassical Crystallization Kinetics of Aspartic Acid in Water

All experimental procedures discussed could be treated as a screening tool for probing the existence of molecular association among the chiral molecules and the solvent system. The molecular association phases of a racemic conglomerate solution (CS) and a racemic compound solution (RCS), and the templating effect of aspartic acid solid surface were observed to minimize the chance of redissolving racemic conglomerate and racemic compound aspartic acid in water and reforming an RCS in crossovers experiments. Only 1 %wt% of l‐aspartic acid was adequate enough to induce a transformation from a racemic compound aspartic acid to a racemic conglomerate aspartic acid. This would make the propagation of biochirality more feasible and sound. However, tetrapeptide, (l‐aspartic acid)₄, failed to induce enantioseparation as templates purely by crystallization. Nonclassical crystallization theory was needed to take into account the existence of a CS. Fundamental parameters of the crystallization kinetics such as the induction time, interfacial energy, Gibbs energetic barrier, nucleation rate, and critical size of stable nuclei of: (i) racemic compound aspartic acid, (ii) racemic compound aspartic acid seeded with 1 %wt% l‐aspartic acid, (iii) racemic conglomerate aspartic acid, and (iv) l‐aspartic acid were evaluated and compared with different initial supersaturation ratios. Morphological studies of crystals grown from the crystallization kinetics were also carried out.Chirality 25:768–779, 2013. © 2013 Wiley Periodicals, Inc.     

八角茴香油溶剂结晶分离的研究

对八角茴香油（ａｎｉｓｅ ｏｉｌ）在三种溶剂中的结晶分离过程进行了研究,考察添加剂对结晶析出的影响,并用溶解度参数理论阐明溶剂的性质与结晶的关系,结果表明,乙醇,甲醇的结晶分离性能较为理想,而丙酮较差;添加剂能显著提高结晶产物的收率,但对结晶产物的纯度有一定的负面影响。     

Evidence for composite cost functions in arm movement planning: an inverse optimal control approach

An important issue in motor control is understanding the basic principles underlying the accomplishment of natural movements. According to optimal control theory, the problem can be stated in these terms: what cost function do we optimize to coordinate the many more degrees of freedom than necessary to fulfill a specific motor goal? This question has not received a final answer yet, since what is optimized partly depends on the requirements of the task. Many cost functions were proposed in the past, and most of them were found to be in agreement with experimental data. Therefore, the actual principles on which the brain relies to achieve a certain motor behavior are still unclear. Existing results might suggest that movements are not the results of the minimization of single but rather of composite cost functions. In order to better clarify this last point, we consider an innovative experimental paradigm characterized by arm reaching with target redundancy. Within this framework, we make use of an inverse optimal control technique to automatically infer the (combination of) optimality criteria that best fit the experimental data. Results show that the subjects exhibited a consistent behavior during each experimental condition, even though the target point was not prescribed in advance. Inverse and direct optimal control together reveal that the average arm trajectories were best replicated when optimizing the combination of two cost functions, nominally a mix between the absolute work of torques and the integrated squared joint acceleration. Our results thus support the cost combination hypothesis and demonstrate that the recorded movements were closely linked to the combination of two complementary functions related to mechanical energy expenditure and joint-level smoothness.     

Influence of low-temperature nucleation on the crystallization process of poly(L-lactide)

The crystallization kinetics of poly(l-lactide), PLLA, is slow enough to allow a quasi-amorphous polymer to be obtained at low temperature simply by quenching from the melt. The PLLA crystallization process was followed by differential scanning calorimetry and optical microscopy after nucleation isothermal treatments at temperatures just below (53 degrees C) and just above (73 degrees C) the glass transition temperature. The crystallization exotherm shown in the heating thermograms shifts toward lower temperatures as the annealing time at 73 degrees C increases. The same effect is shown to a lesser extent when the sample nucleates at 53 degrees C, showing the ability to nucleate in the glassy state, already shown in other polymers. The shape of the DSC thermograms is modeled by using Avrami's theory and allows an estimation of the number of crystallization germs formed. The results of optical microscopy are converted to thermograms by evaluating the average gray level of the image recorded in transmission mode as a function of temperature and calculating its temperature derivative. The shape of such optical thermograms is quite similar to that of the DSC traces but shows some peculiarities after long nucleation treatments. Atomic force microscopy was used to analyze the crystal morphology and is an additional proof of the effect of nucleation in the glassy state. The crystalline morphology observed in samples crystallized after nucleation in the glassy state is qualitatively different from that of samples nucleated above the glass transition temperature, and the number of crystals seems to be much greater than what could be expected from the crystallization kinetics.