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Crystals of thaumatin and of turkey and hen lysozyme, prepared in a batch at 293 K under hydrostatic pressures in the 0.1–150 MPa range, have been analyzed to investigate how this parameter influences crystallization and if depressurization introduces defects in the crystalline lattice. The X-ray diffraction properties of depressurized crystals have been compared with those of unpressurized control crystals prepared under otherwise identical conditions at atmospheric pressure (0.1 MPa). Independently of pressure, the crystals of each protein belong to the same space group and have cell parameters identical to those of the controls. Their diffraction limit is more dependent upon crystal volume than upon pressure. Crystal mosaicity, expressed as the full-width at half-maximum w of the Bragg reflection profile, was used to quantify the defects in the lattice. While the quality of lysozyme crystals deteriorates as pressure increases, that of tetragonal thaumatin crystals becomes more homogeneous. The first result correlates with the apparition of cleavages oriented perpendicularily to the crystal's c -axis. The second one is interpreted by a gradual selection of well-formed nuclei. The 3D structure of thaumatin in a crystal that was grown under a pressure of 150 MPa and returned to atmospheric pressure was solved. The fold of the polypeptide chain and the distribution of water molecules was compared with those in a reference crystal grown at 0.1 MPa.The results suggest that the crystal lattice is elastic. A possible application of pressure to the preparation of high quality protein crystals is discussed in the light of these results.

A quasi-planar X-ray study has been done to assess the quality of crystals of pure thaumatin and of hen egg-white lysozyme as well as of lysozyme which was intentionally contaminated by structurally unrelated (ovalbumin and conalbumin) or related (turkey egg-white lysozyme) macromolecules. To investigate the behavior of different growth sectors, we have chosen crystals exhibiting well defined habit, namely crystals grown either in agarose gel or in silica gel. The main defect evidenced is a misorientation originating at the level of the nucleus: the parts grown in the +c and −c directions seem to be individuals twisted always clockwise with respect to the growth direction. The measured lattice plane tilt increases up to 3.5 min of arc when the contaminant content increases. In addition to this defect, we could measure for lysozyme relative lattice parameters differences (Δd/d) of the order of 10−4 between prismatic and pyramidal growth sectors. All these defects do not seem to have a major influence on the resolution limit but they have consequences on optical properties.

The temperature dependence of lysozyme solubility has been measured in light and heavy water in KCl and in NaNO3 at pH=4.5. The solubilities have been determined with the static light device previously developed to determine the temperature dependence of lysozyme solubility in NaCl. In the present work, we show that the solubility curves in light and heavy water are shifted by 7.2°C in both KCl and NaNO3, as previously observed with NaCl. This temperature shift is the same, which is found between the temperature dependence of density and of other physical quantities of heavy and light water. These new results evidence a general trend of how structural aspects of bulk water can have a profound impact on the properties of biological systems.

Quasi-planar wave reflection profile and X-ray topography studies have been done to characterize the mosaicity of solution- and gel-grown crystals of three proteins, turkey egg-white (TEW) lysozyme, thaumatin, and a bacterial aspartyl-tRNA synthetase (AspRS) as well as of one virus, tomato bushy stunt virus (TBSV). These materials are representative of a large range of molecular weight, overall particle shapes, crystals habits, packings, and solvent contents. Measurements of the full-width at half-maximum (FWHM) of reflections show that these different crystals have all a weak mosaicity. Topographs display the same features as those of the well-studied hen egg-white (HEW) lysozyme crystals: misorientation generated at the seed level for TEW lysozyme or thaumatin crystals and/or strains at growth sector boundaries for AspRS crystals. No growth defects are evidenced for TBSV crystals. For the study of crystals diffracting at lower resolution (AspRS and virus), a less absorbant sample holder, which facilitates crystal positioning in the X-ray beam, has been developed. The results obtained for solution- and gel-grown crystals do not show important differences. However, for TEW lysozyme and thaumatin crystals, one notices a larger dispersion of results in the solution case and an overall tendency for improved reproducibility of quality for gel-grown crystals.

The crystalline quality of hen egg white lysozyme (HEWL) crystals grown in agarose gels and in silica gels has been characterized by measuring resolution and mosaic spread. These crystals have been compared to solution grown ones. A quasi-plane-wave X-ray topography study has also been done on some crystals. The study concerns triclinic, monoclinic, orthorhombic and tetragonal forms of HEWL. One observes that the resolution is not really changed by gelling the growth medium, even for rather high gel contents (agarose 0.5% wt/wt). On the contrary, mosaicity, characterized through reflection profile recordings allows to differentiate crystals grown by different techniques: agarose gel grown crystals are, on average, better than solution grown ones but the best crystals are obtained in silica gel. X-ray topography confirms this result.

In order to understand why hen egg white lysozyme crystals nucleate more easily in agarose gel than in gel-free solutions, we have made a small angle neutron scattering study of gelled and gel-free lysozyme solutions for different supersaturation conditions by varying the temperature, protein and salt (NaCl) concentrations. For higher supersaturations, some kinetics experiments have been done. We have analysed the SANS signal as resulting from two contributions. A first contribution is due to free protein monomers. It shows that concentration and interactions are identical in gel and gel-free media. In this respect, the gel is neutral and supersaturation is not changed. A second contribution is attributed to lysozyme clusters of several tens to several hundreds of nanometers. This contribution increases by increasing supersaturation and, for a given supersaturation, is noticeably stronger in gel than in solution. This effect being not observed when the gel is not set (agarose sol), it has been related to the mechanical properties of the medium. These considerations lead us to propose a link between clustering and nucleation, namely, nucleation would be built out of some of these clusters.

We have determined the temperature dependence of the solubility of lysozyme in light water, heavy water and in a mixture 50% H 2 O 50% D 2 O for various NaCl contents at a given pH. For the solubility measurements, we have used a static light device that we have developed in the laboratory. We have found that the curves representing lysozyme solubility in light and heavy water are shifted by 7.2°C, the difference in the temperature of maximum density of both types of water. As the density of light and heavy water are similarly shifted by 7.2°C, we have plotted master curves, for a given NaCl concentration, of lysozyme solubility as a function of water density. The solubility data, in the 0.3–0.6 M NaCl range, show an equivalence between the effect of 0.1 M NaCl and the isotopic substitution of light water by heavy water. We propose that this equivalence could be explained by the same kind of influence of each effect on the water structure.

Measurements of refractive index as a function of time have been done in supersaturated lysozyme solutions. They reveal that the refractive index decreases in free solutions and increases in gelled solutions during prenucleation. These results are interpreted assuming molecular associations that sediment in free solution and levitate in gel medium.

Gel growth of 2-amino-5-nitropyridinium dihydrogen phosphate (2A5NPDP) has been followed first by optical micrography, which allows to determine growth rates in given directions ([001] and [100]), and second by holographic interferometry, which allows to characterize the evolution of the growth medium through the calculations, at every time, of the supersaturations at the crystal-solution interface. Two growth conditions obtained by varying the solvent concentration have been tested. The growth mechanism appears to be the same governed by a slow surface integration and a slow diffusion of matter.

A series of solution and gel growth experiments have been done to obtain hen egg white lysozyme (HEWL) crystals in media contaminated by a parent molecule, turkey egg white lysozyme (TEWL) or TEWL crystals from media contaminated by HEWL. In both cases, such a contamination causes nucleation inhibition and morphology changes. Evolutions of nucleation and morphology as a function of contaminant percentage show that the contaminant percentage is efficient on nucleation at low percentages ( 20%). This suggests that the contaminant exists in solution as different species probably oligomers, whose action is different for prenucleation and growth processes. Gel media extend the domain for which usable monocrystals are obtained from 50% contaminant (case of solution) to 60% (case of agarose gel) or 70% (case of silica gel).

2-Amino-5-nitropyridinium dihydrogen phosphate (2A5NPDP) is an organomineral material presenting interesting quadratic optical nonlinear properties, in particular towards second harmonic generation. Single crystals have been grown by spontaneous nucleation in tetramethoxysilane gel by slow cooling. X-ray characterization provides evidence of their good crystalline quality. Second harmonic generation experiments at 1.34 μm have confirmed the efficiency of these crystals and permitted inferring the d15 and d24 coefficients from the susceptibility tensor.

Two populations of aggregates are generally indentified in supersaturated solutions of biological macromolecules: small aggregates of a size which is less than 5 nm and large aggregates, the largest of which are at least one order of magnitude bigger. In order to understand the role played by the microporous network of a gel in the growth and behaviour of these different species in the prenucleation period, an in situ observation of nucleation has been carried out using either free solutions or solutions trapped in agarose gels. In a previous study, free solutions were investigated by small-angle neutron scattering (SANS) to identify the small aggregates. Optical observations, made under the same conditions, revealed the formation of an amorphous precipitate which disappeared at the end of the experiment. The sedimentation of this phase, which occurs in free solution but never occurs in gelled solution, depletes the solution bulk and this could explain why the nucleation density is higher in agarose gel than in free solution. The case of silica gel, the behaviour of which is completely different with respect to nucleation, will be discussed.

A procedure which allows an investigator to supply a crystal with fresh mother material without inducing significant growth defects is described. This technique requires that the crystal is grown in a gelled hanging or sitting drop. An example concerning a model macromolecule, hen egg-white lysozyme, is given. Extension of this procedure to other macromolecules is discussed.

In order to investigate how macromolecular aggregation proceeds for obtaining nucleation and crystal growth, a series of hen egg white (HEW) lysozyme solutions representative of a large supersaturation range have been investigated by small angle neutron scattering (SANS). In all these solutions, the signal well corresponds to species of radii of gyration Rg below 50 A. Moreover, the effective values Rg are compatible with a rather monodisperse distribution of species the size of which increases when the supersaturation increases; for example, saturated solutions correspond to dimer populations. The largest size which has been identified corresponds to octomers which seems a limit beyond which nucleation and growth occur. The growth units are larger than dimers and probably correspond to tetramers or octomers. SANS allows one to study kinetic aspects. We observe that when a given supersaturation is quickly established, the radius of gyration increases with time. In the light of these results, it appears that SANS affords a powerful tool to study aggregation phenomena occurring in the metastable zone.

International audience; Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Convection effects can be prevented by gelling the hanging drops used in protein crystal growth. An exploratory study has been made on a model material, hen egg white lysozyme, growing in agarose gel. In that case, it is observed that using gel promotes nucleation.

Silicon tetramethoxide monomers are dissolved in water, hydrolyzed, and condensed to form a mesh of siloxane polymers. The growth and aging of this gel are examined through light and X-ray scattering. From interferences at short distances, the fractal dimension, D, of the growing polymers is measured. From interferences at large distances, the mesh size, ξ, of the resulting network is measured, which scales with concentration: ξ is 400 A when the volume fraction of polymer is φ = 0.04, and 10 000 A when φ is 0.004. The scaling laws which are determined from the final state of the gel differ from those which describe its growth patterns.

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75–100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with ∼20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

Crystals of thaumatin and lysozyme from turkey and hen egg-white have been prepared under hydrostatic pressures in the 0.1-220 MPa range in batch at 20°C in agarose gel. For the three proteins, the number of crystals increases with pressure. For thaumatin and hen lysozyme, crystal length decreases while for turkey lysozyme it increases. The solubility of the first protein decreases, whereas the solubility of both lysozymes increases. In all cases, the relationship between solubility and pressure is linear. The crystallization volume ΔV is -11 cm3 mol−1 for thaumatin, +15 cm3 mol−1 for turkey lysozyme, and +3 cm3 mol−1 for hen lysozyme. For the lysozymes, the values are explained by pressure-dependent conformational changes. The crystals have been analyzed by x-ray diffraction methods. Independently of pressure, the crystals of each protein belong to the same space group. Crystal mosaicity has also been determined. The quality of the lysozyme crystals deteriorates as pressure increases, however thaumatin crystals become more homogeneous. The first result correlates with the apparition of cleavages oriented perpendicularly to the crystal’s c axis. The second one is interpreted by a gradual selection of well-formed nuclei. A potential application of pressure for the preparation of high quality protein crystals is discussed in the light of these results.

NH4H2PO4 (ADP) crystals have been grown in a tetramethoxysilane gel by temperature decrease. Nucleation and growth have been followed by interferometric holography which has allowed to determine the supersaturation so that the growth proceeds by a bidimensional mechanism without generation of dislocations.

A plane wave imaging technique has been applied to the characterization of growth defects in Ba(NO 3 ) 2 and Sr(NO 3 ) 2 crystals. Precise values of lattice parameter differences between cubic and octahedral sectors have been obtained in the case of Sr(NO 3 ) 2 . For Ba(NO 3 ) 2 , plane wave observation confirms the crystalline perfection revealed by Lang topography.

ADP crystals doped with chromium were obtained by temperature decrease of a saturated aqueous solution either stirred (solution-growth) or gelled (gel-growth). Characterization of the doping effect was made by atomic absorption, optical observations (tapering angle, colour) and X-ray Lang topography. The global incorporation of chromium is found to be higher in the gel-grown crystals; however, the lattice strains associated with the relative segregation of chromium in the prismatic and pyramidal sectors are reduced.

Gypsum (CaSO4·2H2O2) crystals which are large and good enough to be characterized by X-ray topography have been grown from solution and from gel in order to study their twin growth.

An X-ray topographic study of potassium bichromate crystals shows that the crystalline quality is improved by increasing the growth temperature. Nearly perfect crystals can be obtained. In that case, it is observed that the presence of a few dislocations strongly influences the growth habit.

Single crystals of CaHPO 4 ·2 H 2 O and PbHPO 4 were grown in silica gel and tetramethoxysilane gels with different concentrations of the gel forming agent and different pH values. At a high growth rate of several mm per day the crystals exhibit a good quality. Low density gels yield at low pH values almost perfect crystals.

X-ray topographs from hyfrothermally grown single crystals of calcite have given evidence for misorientations between growth sectors. Furthermore, strained areas revealed by cleavage show mismatching in a same growth sector between two parts, one grown from the center of the face, the other from the edges. According to the value of local supersaturation, one of the growth processes prevails over the other.

The characteristics of gel growth are compared to those of classical solution growth, showing the advantages and drawbacks of different growth media. The main techniques used to grow crystals in gels are illustrated in the case of hen egg white lysozyme crystals.

Facilities for topographic studies using Synchrotron Radiation Sources are reviewed and discussed as for growth defect characterization. Two types of studies are most used: kinetic studies performed by using white beam topography and lattice parameter mapping obtained by plane wave topography.

NH4H2PO4 crystals moderately doped with trivalent chromium (30–130 ppma in the mother solution) were obtained either in a stirred solution (solution growth) or in a tetramethoxysilane gel (gel growth). Characterization of the doping effect was made by means of traverse or section X-ray Lang topography, plane-wave synchrotron topography and secondary ion mass spectrometry in order to have direct correlation between impurity distribution and lattice distortion. It is shown that the preferential chromium segregation in the prismatic growth sectors is less pronounced for gel-grown than for solution-grown crystals and correlatively the lattice parameter variation Δcc is smaller. Strain distribution between prismatic and pyramidal growth sectors also differ: in gel-grown crystals, the lattice displacement gradient is localized at the growth sector boundary whereas, in solution-grown crystals, it is expanding through the bulk of the prismatic sectors.

Large single crystals of Na2CoGeO4 were grown under hydrothermal conditions. Two methods are used: crystallization on seed and spontaneous growth in temperature gradient conditions. The comparison of the defect distribution shows the best quality for crystals obtained with the most rapid growth rates.

Various experimental methods have been used in order to study the mass transport of NaCl in tetramethoxysilane (TMS) gels. The concentration of NaCl has been measured either through refractive index (“holographic interferometry”) or electrochemical potential (“extraction method” and “diffusion through a gel”). The values of the diffusion coefficient of NaCl in TMS gels have been deduced.

KDP crystals grown from boiling solutions present well developed prismatic sectors and show a good crystalline quality while being obtained at high growth rates.

Lead hydrogen phosphate (LHP) and lead nitrophosphate (LNP) crystals have been grown by counterdiffusion of lead nitrate and phosphoric acid solutions either in a gelled medium or in a free medium under the reduced gravity environment of Spacelab. Characterization of grown crystals allows to conclude that diffusive regimes are obtained in both xases. Dielectric measurements reveal significant differences which can be correlated with the crystalline quality.

Growth defects in single crystals of potash alum grown with various conditions have been examinated by X-ray topography. An asymmetrical distribution of defects can be related to the direction of the flux. From static solutions, large defect free areas can be obtained in sectors corresponding to slow faces.

Water soluble crystals such as ADP and KDP have been grown in TMS gels by different techniques. A study of growth defects by X-ray topography shows that these gel grown crystals obtained by a very simple technique can be compared to crystals grown from solutions using elaborated systems.

In order to elucidate how defects appear during growth, known perturbations have been generated and the nature of the defects they induced was investigated by means of X-ray topograohy. They are essentially growth bands, inclusions and dislocations. The growth rates could be determined and it was shown that the presence of dislocations increased the growth rate inasmuch as they have a strong screw component. Furthermore, nearly dislocation free and perfect crystal has been observed on “S” type faces, according to Hartman's classification.

Potash alum crystals grown in solution with and without polyethylene oxide traces are compared: the crystalline quality is improved in the first case. An interpretation is attempted.

Silica gels were used as a media for crystal growth of hen egg white lysozyme crystals. Using small angle neutron scattering, gel-free solutions have been compared to gelled samples in the pre-nucleation period and in the growth period. Characterisation of the protein solution trapped in the gel network shows an adsorption of protein on the gel surface through electrostatic and most probably through hydrogen bonding interactions. The consecutive decrease of concentration is consistent with the decrease of the nucleation rate with respect to gel-free solutions. By modification of the gel surface, we succeeded in increasing the number of nuclei. Growth occurs by incorporation of free molecules and in a second stage, by incorporation of molecules which are progressively desorbed. This provides a relatively constant concentration of the free molecules.

A study of lysozyme-lysozyme interactions by small angle neutron scattering (SANS) as a function of temperature in D2O and in H2O is presented. The interactions are analysed in terms of second virial coefficient. The most remarkable result is that the difference between the temperature dependencies of this quantity in D2O and H2O seems similar to the one we have previously observed for lysozyme solubility in these two solvents. These physicochemical quantities in D2O, the second virial coefficient and solubility, are indeed shifted by approximately 7.2°C relatively to the ones in H2O. This temperature shift, 72.°C, corresponds to the difference of the temperatures of maximum density of H2O (4°C) and D2O (11.2°C). The interactions are modelled by the DLVO potential, which includes an attractive van der Waals component, proportional to the Hamaker constant AH and a repulsive screened electrostatic term, proportional to the net charge of the protein Zp. The attractive component seems to phenomenologically account for effects due to the water structure, as the Hamaker constant AH also appears to be shifted by about 7.2°C.

Nearly perfect brushite crystals can be grown quickly in gels as checked by X-ray topography.

Discrepancies in lattice parameter determinations of lead phosphate crystals are discussed in terms of crystal growth.