Formation and Growth of Tetragonal Lysozyme Crystals at Some

Feb 4, 2009 - The influence of a second flat glass plate as well as some differences in the supersaturation is shown; the four {101} pyramidal faces v...
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CRYSTAL GROWTH & DESIGN

Formation and Growth of Tetragonal Lysozyme Crystals at Some Boundary Conditions

2009 VOL. 9, NO. 3 1312–1317

Daniela S. Tsekova* Department of Organic Chemistry, UniVersity of Chemical Technology and Metallurgy, 8 St. Kliment Ohridski BouleVard, 1756 Sofia, Bulgaria ReceiVed April 8, 2008; ReVised Manuscript ReceiVed December 24, 2008

ABSTRACT: Formation and growth of tetragonal lysozyme crystals at the interface of protein solution with glass substrate have been studied. Morphology of crystals formed and grown on a flat glass surface are given and discussed. The influence of a second flat glass plate as well as some differences in the supersaturation is shown; the four {101} pyramidal faces vanish, giving a place of a hemisphere or a “new” {001} face.

1. Introduction Hen egg white lysozyme (HEWL) is an easy to crystallize protein. It has been used by many researchers as a model substance for investigation of some general crystalline properties of proteins.1-3 Six different symmetries of its crystals have been described until now,4 the most examined of which is the tetragonal syngony (Figure 1), formed by the association of a tetragonal {110} prism and a {101} tetragonal bipyramid.5 Its elementary cell (P43212, a ) b ) 7.91 nm, c ) 3.79 nm) is a tetragonal Bravais lattice, rectangular prism with a square base, and a height different from the length of the square (Figure 1B). Crystallization of proteins is a function of many parameters of the solution. The protein solution behavior often is given by a two-dimensional phase diagram that displays variation of two parameters, keeping all others constant. Such a phase diagram shows the state of a material, solution or solid at different sets of conditions,6 and a mainly illustrative one, including some points derived from experiments described here, is presented bellow. This paper covers data for morphological features of tetragonal crystals from HEWL formed and grown on a flat glass substrate, as well as the case of a second glass plate restricting the space of growth. All experiments were performed at a constant lysosyme concentration and buffer; only the temperature, concentration of NaCl, and the thickness of solution layer were varied. The morphology of crystals heterogeneously nucleated on flat substrates is determined by the interaction between molecules and substrate. Observations of kinetic roughening at low temperatures and high supersaturations as well as new data for habitus changes at space limitation are reported.

2. Theoretical Base This work is devoted to formation and growth of tetragonal lysozyme crystals in a two-dimensional cell, constructed by two parallel flat glass plates (see Figure 3 and Experimental Procedures, Materials and Methods). That is why some theoretical considerations for the influence of a foreign flat surface on the formation and growth of crystals are schematically outlined here. It is well-known that foreign substrates enhance the formation of nuclei.7 At given sets of conditions, for example, * To whom correspondence should be addressed. Phone: +(359)28163418; e-mail: [email protected].

at the same supersaturation, nucleation occurs preferentially on the substrate surface, rather than in the homogeneous phase, and critical nuclei formed on a flat foreign surface possess a reduced volume and reduced number of faces than those formed in solution. The causes are of a thermodynamic nature and due to the interactions between substrate and molecules forming the new phase. According to Kaischew7a,c and Mutafschiev,7b the relation between volumes of the critical nuclei formed on substrate, Vsj, and the homogeneously formed one, V, is equal to the relation between the corresponding nucleation works (energies necessary for nuclei formation) on the substrate ∆Asj, and for one nucleated in a homogeneous way, ∆A. It also depends on the ratio Ψadh-j/Ψj, where Ψadh-j is the adhesion energy between the substrate and lysozyme molecule laying on it with j side, and Ψj is the cohesion energy in the crystal in the same crystallographic direction; it is expressed by eq 1.

(

Ψadh-j Vsj ∆Asj ) ) 1V ∆A Ψj

)

(1)

2D visualization of the shapes and sizes of critical clusters (nuclei) of tetragonal lysozyme crystals, based on an approach proposed by Kaischew7c that concerns crystallization of small molecules, is given in Figure 2. Here the elementary cell (see Figure 1B), presented by a rectangle with sizes a and c, builds critical nuclei (again in a two-dimensional model), in which sizes A(A1) and C(C1) are proportional to a and c. Two of the simplest ways to dispose of this cell on the substrate are illustrated: when ordered in the critical nuclei elementary cells lay on the substrate with a side (see Figure 2, case 2) and when c side adheres to the substrate (see Figure 2, case 3). If the homogeneously formed critical nucleus has sizes A and C, then those formed on the substrate will differ in size, for example, with lengths A, C1 when A side contacts the substrate, and A1, C when C contacts the substrate (Figure 2). In the case that Ψadh-a and Ψadh-c have close values, both orientations of the critical nuclei presented on the Figure 2 as cases 2 and 3 will be observed with nearly equal probabilities. If the difference is great enough, then the orientation of the critical nucleus with higher Ψadh-j (j ) a or c) will grow dominantly as the corresponding ∆Asj will be lower. Once the energetic threshold is overcome, the critical nuclei continue to grow. The growth shape of a crystal is determined by the shape of its critical nucleus and by the rates of growth of existing faces in this shape.7e Substrates influencing crystal nucleation are two types from the crystallographic point of view - structured and structureless.

10.1021/cg800361v CCC: $40.75  2009 American Chemical Society Published on Web 02/04/2009

Growth of Tetragonal Lysozyme Crystals

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Figure 1. Tetragonal lysozyme crystals. (A) Schematical ilustration; (B) elementary cell a ) b * c; R ) β ) γ ) 90°; (C) lysozyme crystal obtained from 25 mg/mL lysozyme, 1 M NaCl in 50 mM sodium acetate buffer, pH 4.5, T ) 20 °C.

3. Experimental Procedures

Figure 2. 2D elementary cell with dimensions a and c and three critical clusters built by it are schematically drawn. Clusters are nucleated homogeneously in the bulk of the mother phase (case 1) and on the foreign surface laying on it in two different orientations (cases 2 and 3). Note that A or A1 is proportional to a, and C or C1 is proportional to c.

Figure 3. Quasi two-dimensional cell: 1 - two flat, optically polished, and welded in parallel position circle glass plates; 2 - removable caps; 3 - thermocouple.

Structured ones often induce crystallization by provoking epitaxy. Glass having an amorphous 3D network of Si-O-Si-O bonds, interrupted in places with metal ions, is representative of the structureless substrates. In aqua solutions Si-O- segments are available on its surface, due to processes of dissociation. This leads to distribution of mobile negative charges along the glass surface, and in this meaning electrostatic potential takes place on the interface glass-water solution that contributes to the processes on this boundary. Protein molecules as crystal building blocks differ to some extent from the small ones (small molecules or atoms) and two main differences can be pointed out: the sizes and the complexity of the molecules from a chemical point of view. Sizes: Proteins are molecules that contain a large number of atoms. If we consider that a typical atom is on the order of 2 Å in diameter and a lysozyme molecule has a 30 Å diameter, it is easy to find that the area of both projections over the substrate differ more than 200 times. Complexity: The great diversity of functional groups of atoms distributed on the surface of protein molecule makes it nonuniform from a chemical point of view as well as of charge distribution and because of that its incorporation into the crystal lattice needs an exact orientation.8 That is why it is expected that the orientations of ordered on the substrate HEWL molecules, building the critical nuclei, unambiguously will determine the orientation of the crystal grown.

3.1. Materials and Methods. Sigma, 3× crystallized HEWL substance was used in the experimental work. All crystallization experiments were performed in batch mode by adding equal volumes of protein solution (50 mg/mL lysozyme solution in 50 mM sodium acetate at pH 4.5) and of the precipitating agent (precipitant NaCl in the same buffer, which concentration varied: 0.6, 0.8, 1.0, and 2.0 M), both prepared at room temperature. Prior to crystallization, all solutions were filtered through 0.22 µm syringe filters (Millipore). Experiments were accomplished in quasi two-dimensional glass cells (Figure 3).9 This cell was constructed from two circle glass plates with diameter 25 mm, welded in exactly parallel positions at distances of 500, 200, or 65 µm between the upper and lower substrates, which was the thickness of the solution layer. This system has two openings, connected with glass tubules and supplied with polished stoppers. Used for this purpose glass substrates were optically polished to achieve flatness in the range of nanometers. Such a constructed cell provides a large surface area with a small amount of solution. All of the experiments described here were carried out in the temperature range from -2 to 23 °C. As the lysozyme solubility is strongly dependent on the temperature, a specific thermal regime of temperature variations, so-called thermal pulse technique, was elaborated in order to nucleate HEWL crystals at a high enough supersaturation and then to grow them at lower supersaturation.3,9 The supersaturation here was defined as σ ) ln(C/Csat), where C is the actual concentration and Csat is the equilibrium one. Although the changes in the supersaturations here were achieved by variations of NaCl concentration and temperature, in all the experiments σ was calculated using data for C and Csat of the lysozyme. (The determination of Csat at HEWL by interferometric measurements is explained in detail by Sazaki et al.10) Different temperature pulses were applied depending on the solute concentrations and target supersaturations. Pt-PtRhthermocouple was used for direct temperature measurements inside the working glass cell (see Figure 3, element 3). HEWL crystals were observed microscopically, both in transmission and reflection mode using an interference optical microscope “Peraval Interphaco” Carl Zeiss, Jena. Interferometric technique, based on the Mach-Zehnder scheme in transmission microscopy, was applied for better contrast and measurement of crystal forms. 3.2. Experimental Set Up. Experimental work ranged over two main directions: The first one was the influence of bare glass flat substrate on the morphology of lysozome crystals that nucleate and grow on its surface. The role of a second (restricting the space of growth) plate, placed parallel to the first one, on crystal morphology also was followed. The other direction was to establish and to use the influence of some variations of the NaCl concentrations and temperature changes. The HEWL concentration was kept constant, 25 mg/mL, in all final working solutions. The solvent for all experiments also had constant values, namely, 50 mM sodium acetate at pH 4.5. NaCl concentration was varied as 0.3 M, 0.4 M, 0.5 M, and 1 M. Temperature variations were in the range -2 to 23 °C. Almost all experiments were performed by applying the temperature pulse technique. Its application consists of lowering the temperature, which leads to an increase of the supersaturation of the solution to values inside the nucleation zone, for a given time, and then raising the temperature back, to push the supersaturation out of nucleation zone, but enough for growth of the nucleated crystals, that is, the growth zone (see Figure 5).

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Tsekova

Figure 6. Growth of lysozyme crystal; (a, b) rounded shapes obtained at very high supersaturations (T ) 0 °C, σ ∼ 3.6) and observed immediately after shifting to room temperature. (c, d) Faceted shapes with clearly visible edges between {101} faces, obtained ∼10-15 min later at lower supersaturation (T ) 22 °C, σ ∼ 0.5).

Figure 4. Tetragonal HEWL crystals nucleated and grown on flat glass surface. (A) Schematically depicted general types of the observed HEWL crystal orientations; (B) optical microscope images of lysozyme crystals, observed in the described experiments.

Figure 5. Phase diagram in coordinates: temperature [°C] and NaCl [M] concentration. Lysozyme concentration is 25 mg/mL in 50 mM sodium acetate buffer, pH 4.5. Solubility curve separates undersaturated from the supersaturated region; the supersaturated one is divided into metastable, nucleation, and precipitation zones.

4. Results 4.1. Substrate Effect. Crystals grown in quasi two-dimensional glass cells especially at lower supersaturations did not possess all of the faces typical for a well-developed bulk-grown crystal, but usually only part of them appeared. Depending on the crystal plane just laying on the substrate, the surface nucleated lysozyme crystals grew with different orientations,3,9a which are presented in Figure 4. The probabilities of distribution of the crystal orientations were found to depend on all other conditions of the experiment (solute concentrations, temperature, supersaturation) revealing that (d) and (f) had higher frequencies for the bare glass surface.9a 4.2. Effect of Salt Concentration. All the experiments were performed with solutions containing 25 mg/mL lysozyme in 50 mM sodium acetate buffer, pH 4.5, in quasi two-dimensional glass cells. Keeping the concentration of lysosyme and buffer constant, variations in the supersaturation were achieved by the salt (NaCl) concentration and temperature changes. The NaCl

concentration was varied as 1, 0.5, 0.4, and 0.3 M. The temperature interval that was applied in order to investigate the crystallization behavior of the lysozyme was -2 to 23 °C. A phase diagram with both variables temperature and NaCl concentration is presented in Figure 5. Some points from this diagram were defined in the experiments described here; partial information was used from other literature sources.6 1 M NaCl. This concentration allows bulk nucleation at room temperature (20 °C). Crystals obtained from this solution possess all the faces of the full crystallographic shape, shown in Figure 1A. The experimentally observed one is given in Figure 1C. Fully developed crystals were grown only in cells with 500 µm distance between both glass plates. In the thinner cells such crystals grew more or less deformed because of the lack of space. 0.5 M NaCl. In these solutions crystals could be nucleated up to 18 °C. They were grown at room temperatures from 18 to 23 °C. The obtained protein crystals were well shaped and stably grown. As the solution with this concentration at t e 0 °C is highly supersaturated (σ g 3.6), then the process of nuclei formation is extremely rapid. A cell with such solution was left at 0 °C up to 2 h then put on the microscopic stage at 22 °C and observed immediately. The first detected HEWL crystals were not polygonized. As it is seen from the photos (Figure 6a,b), their initial form includes a semisphere in the place of the expected four {101} pyramidal faces, and the only observed edges of these rounded crystals were around the semisphere. During the subsequent growth in the next 10-15 min, crystals were gradually polygonized at the realized lower supersaturation (σ ∼ 0.5 at 22 °C). The edges between {101} and {110} faces continuously appeared (Figure 6c,d) and the end-growth form was faceted by them, thus obtaining the typical habitus of tetragonal HEWL crystal (Figure 1C). 0.4 M NaCl. Crystals were nucleated at temperatures lower than 8 °C (σ g 1.6). They were well shaped and grew in a stable way until 18 °C. At higher temperatures, they start dissolving. 0.3 M NaCl. This salt concentration is very low and the nucleation here could be expected at low enough temperatutes. Indeed, crystals nucleation was noticed only at very low temperatures, namely, below 0 °C (σ ∼ 1.6). Further growth was possible only under relatively low temperatures, that is, ∼4-5 °C. At higher temperatures these crystals dissolved easily. At these conditions all crystals that grew with the c-axes perpendicular or declined to the substrate (corresponding to

Growth of Tetragonal Lysozyme Crystals

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hanging drop in the case of mechanical obstacle and diffusion limitation. Thus, these growth restrictions bring about polyhedral instability.

5. Discussion

Figure 7. {001} face of a prismatic tetragonal lysozyme crystal in quasitwo-dimensional cell (200 µm) obtained from solution: 25 mg/mL HEWL, 0.3 M NaCl in 0.05 M sodium acetate buffer, pH ) 4.5; temperature of formation and growth: -2° to 0 °C. Observation at room temperature, immediately after transfer of the cell to the microscope.

Figure 8. HEWL crystals grown between two parallel glass substrates with a distance of 65 µm; (a) Prismatic tetragonal lysozyme crystal, with terraced {001} plane of HEWL crystal; the highest level (in the middle) is 500 Å; (b) flaws and (c) defect on the plane with restricted material supply. {101} faces are seen on crystal vertices; conditions: 25 mg/mL lysozyme, 0.5 M NaCl in 50 mM sodium acetate buffer, pH 4.5, temperature of nucleation 8 °C, temperature of growth 20 °C, space in the cell - 65 µm.

Figure 4a,d) had unusual crystal morphology. They took the shape of a simple tetragonal prism (Figure 7) and a new {001} face appeared instead of the pyramid formed by the known four {101} faces. In Figure 7 this {001} face seems smooth and cracked and a square with different level can be detected inside (edge length 200 µm). Crystals grown with the c-axes parallel to the substrate revealed a hexagonal shape of the {110} face, for example, 4b and their morphology was not exchanged. 4.3. Growth at Space Limitations. When protein solution with 0.5 M NaCl was placed in the very thin cell with 65 µm distance between both glass plates, the HEWL crystals growing with the c-axes perpendicular to the substrates, that is, with four {101} faces atop (see Figure 4, orientations a and d), again displayed a change in their habitus. Like the case presented above (Figure 7), they took the shape of a simple tetragonal prism with one {001} face on its top, instead of the four {101} pyramidal faces. The square {001} face seemed flat under low microscopic magnifications and the interferometric study showed several concentric square terraces, the highest one in the central part of the face being 40-50 nm high (Figure 8a). HEWL crystals with {110) faces (and c-axes) parallel to the glass plates (Figure 4b) again were growing without any changes in the habitus. The consequent growth of crystals at conditions of such space limitations led to morphological instability.9a When a crystal almost reached the second glass plate, starvation flaws appeared on the crystal termination (Figure 8b,c). Evidently, these flaws are a direct result of the fact that the thin solution layer resting between the crystal and the second glass surface does not permit a sufficient protein supply. In a previous study,11a we have observed the same phenomena with HEWL crystals grown in a

5.1. Orientations of Tetragonal HEWL Crystals Heterogeneously Nucleated on Flat Glass Substrate. Obviously HEWL crystals presented in Figure 4a-f resemble the crystal presented in Figure 1, but intersect with the substrate in six different ways. As it was considered above, heterogeneous nucleation supposes that adherring to the substrate lysozyme molecules build a contact plane between the critical nucleus and the foreign substrate and further incorporation of protein molecules into a growing crystal requires that each molecule ultimately becomes properly oriented and positioned with respect to its neighbors in the crystal.8 We expect that only the faces shaping the nucleus continue to grow. For example, when lysozyme molecules adhere to the substrate so oriented that the formed crystal is with the c-axes perpendicular to the substrate, a shape with only four {101} faces will appear in the direction of growth and crystals with morphology corresponding to Figure 4a will grow. So in the case of nucleation of crystals on the flat substrate the different observed crystal orientations, presented in Figure 4, inherit differently oriented critical nuclei, which form due to the adhesion of the lysozyme molecules to the substrate (depicted in Figure 2). In the case of heterogeneous formation of lysozyme crystals, it was found that some surfaces promoted only one type of orientation.3,12 This fact again can be explained by adhesion of the HEWL molecule in a preferred orientation to a specific surface at a given set of conditions. In a previous work,11b we have described lysozyme crystals grown between two substrates with a distance of 300 µm, applying the sandwich drop technique. They were used in experiments for measurement of the strength necessary to detach crystals from the substrate.11b The main part of the crystals was strong enough stacked to the foreign surface, and forces applied in order to detach such crystals from glass substrate were in the range of 11-14 N cm-2.11b Part of them were stacked to the bottom glass slides, and the rest were stacked to the upper ones, which is an additional evidence for the substrate nucleation (not for sediment crystals). Sanjoh et al.13 investigated heterogeneous nucleation of lysozyme on flat substrates constructed from semiconductor (pSi) covered by thin-film - nSi, SiO2, Si3N4, and Al2O3 unviolated and etched surfaces, in an aqueous electrolyte solution. They found that the surface potential at specific sets of conditions (pH, buffer, and salt concentrations) enhances formation of crystals. Similarly, in experiments described here, at pH ) 4.5, the lysozyme molecule is positively charged and the glass surface carries some unstable negative charges, which most probably leads to electrostatic attractions between the glass substrate and the protein molecules. These interactions obviously play a role in the facilitation of nucleation, as well as in the distributions of crystal orientations. In our previous studies of nucleation of HEWL crystals on bare glass surface, surface covered by poly-L-lysine (PLL) and surface treated by hexamethylsylazane (HMDS) to make it hydrophobic, it was found that critical nuclei formed on bare glass surface (consisting of four molecules) are 2 times smaller than those formed on the other two substrates (eight molecules included).11c This result can be explained with stronger interactions, due to the electrostatic attractions. On the other hand adhesion strength measurements for crystals nucleated and grown on the three surfaces

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show again that in the case of bare glass surface this strength is about 3-4 times higher.11b 5.2. New Face Appearing in a Confined Environment. At solutions of 25 mg/mL lysozyme, 0.5 M NaCl in 50 mM sodium acetate buffer, pH 4.5, temperature pulse at 8 °C, temperature of growth 20 °C, space in the cell, 65 µm, most of the crystals with orientations 4a and 4d possessed prismatic shape. Such a crystal is presented in Figure 8a. The four pyramidal {101} faces are replaced by a {001} face. Here it was observed flat without any visual at the magnifications defects {001} face, having some terraces parallel each to other. As this prismatic shape obtained at the above set of conditions was observed only in the very thin cell - 65 µm, but not in broader ones (200 µm), obviously one of the reasons for its appearance was the narrow space. In a broader cell with space 200 µm, prismatic crystals appear as well, but only at a lower concentration of NaCl and low temperartures. From solutions of 25 mg/mL HEWL, 0.3 M NaCl in 0.05 M sodium acetate buffer, pH ) 4.5, space in the cell of 200 µm, temperature of formation and growth, -2° to 0 °C, all crystals grown with orientations 4a and 4d in a cell with a distance of 200 µm, took a prismatic shape (Figure 7). As at low supersaturations tetragonal lysozyme crystals tend to show an elongated form along [001], it is quite possible that the growth of the {101} faces is hampered due to the presence of the upper glass plates. Moreover, crystals grown with c-axes parallel to the substrate had in their habit {101} faces and displayed morphology similar to that presented in Figure 4b. Probably the reason for the second {001} face appearance in the crystals growing with the c-axes perpendicular to the substrate, which are analogs to those presented in Figure 4a,d, is the same as in the very thin cell with 65 µm spacing restriction of space for growth. In both cases (Figures 7 and 8a) although {001} is flat, additional squarer shaped flat layers are visible. The flatness of the new face suggests that it may exist in the habitus of tetragonal lysozyme crystals and is not a result of instability, but probably at normal conditions it possesses high surface energy and energetically more favorable {101} bypiramidal faces form on it. 5.3. Morphological Defects Development. When a growing crystal reached the second plate of the cell, and continues to grow, mechanical defects, for example, starvation flaws, formed on the plane reaching this glass substrate (Figure 8b,c). It is well-known that the crystal morphology strongly depends on the process of diffusion supply, and the limitation of this supply leads to formation of morphological defects.11a These kinds of defects (Figure 8b,c), which are the results of bad material supply, do not develop on the contact plane crystal/substrate, as the crystal growth there is realized by the incorporation of lysozyme molecules into the kink positions situated also on the interface where the crystal was nucleated. The visual defects (cracks) of {001} faces for the crystal presented in Figure 7 were probably caused by the difference in temperatures of growth (t e 0 °C, σ ∼ 1.6, supersaturated solution) and of taking photography (t ∼ 20 °C, undersaturated solution), which led to great differences in the supersaturation. Probably this undersaturation of the solution and temperature changes caused very fast crushing in the crystals. 5.4. Kinetic Roughening. At very high supersturations rounded shapes were observed where instead of the four pyramidal {101} a semisphere appeared as seen in Figure 6a,b. Lowering the supersaturation (by warming the system) caused formation of {101} faces and edges (Figure 6c,d). The phenomenon of rounded faces developing, known also as “kinetic roughening”, is often observed on growing organic crystals.

Tsekova

Faces that are smooth at equilibrium may become round if they grow at sufficiently large supersaturation.14 Kinetic roughening of tetragonal lysozyme crystal was reported by Gorti at al. as well.15 Crystals presented in Figure 6a,b possessed a rough and rounded surface without any sign of {101} face or edges between them. Consequent growth of such close to spherical patterns at high enough supersaturations led to formation of the crystal’s agglomerations. When the concentration (respectively supersaturation) was increased further, for example, by evaporation, spherulites were formed. Other authors also reported that at higher supersaturations protein solutions tend to form spherulites,16 while at lower concentrations polyhedral crystals were obtained. Pronounced differences in the habitus of the tetragonal lysozyme crystals obtained at different concentrations of NaCl, HEWL, and temperature variations were published from other authors also;1c,12a nevertheless, until now the appearance of the {001} face in the habitus of the tetragonal HEWL crystals was not reported in the literature.

6. Conclusions Lysozyme crystals formed on a flat glass substrate display various orientations which are determined by the orientation of those that initially adhered to the foreign substrate HEWL molecule. More than one final shape of the heterogeneously formed and grown lysozyme crystals were observed (Figure 4) and that points to the fact that the lysozyme molecule under this condition (25 mg/mL lysozyme, 0.5 M NaCl in 50 mM sodium acetate buffer, pH 4.5) can adhere to the used foreign substrate (bare glass here) in more than one favorable orientation. From the experiments conducted, it is obvious that the pyramidal {101} faces are more sensitive to environment changes than the prismatic {110} ones. Under conditions of very high supersaturations, they reveal kinetic roughening and grow as a semisphere and at some conditions of space limitation they disappear giving the place of the “new” {001} face and HEWL crystallizes in a simple tetragonal prism. Until now this habitus of prismatic HEWL crystals has not been reported in the literature. The growth of the flat planes {001} is evidence that this face could exist, but probably it possesses very high surface energy and the formation of {101} bipyramidal faces is favored. When crystal growth is hampered by transport limitation or just exhausted of protein solution, morphological defects develop on these faces of crystals that were also reported before for HEWL crystals grown in hanging drops.11a Acknowledgment. The author expresses acknowledgments to Prof. C. Nanev, supervisor of her Ph.D. Thesis in the Institute of Physical Chemistry “Rostislav Kaischew”, Bulgarian Academy of Sciences, where the experimental part of this work has been carried out.

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