Observation of Clear Images of Dislocations in Protein Crystals by Synchrotron Monochromatic-Beam X-ray Topography† Mayumi Koishi, Naoki Ohya, Yu Mukobayashi, Haruhiko Koizumi, Kenichi Kojima, and Masaru Tachibana*
CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2182–2186
International Graduate School of Arts and Sciences, Yokohama City UniVersity, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan ReceiVed September 27, 2007; ReVised Manuscript ReceiVed September 28, 2007
ABSTRACT: Crystal defects, especially dislocations, in hen egg-white lysozyme crystals that have multiple polymorphisms, for example, tetragonal, orthorhombic, monoclinic forms, etc., were investigated by means of synchrotron monochromatic-beam X-ray topography. The observed topographic images of dislocations were much clearer compared to those of any protein crystals that have been reported so far. It was demonstrated that millimeter-size crystals larger than extinction lengths for X-ray topographic reflections are required to obtain clear images, that is, direct images, for protein crystals. In addition, the weak-beam technique was found to be useful for obtaining clearer images. Straight, curved, and loop-type dislocations were clearly resolved on the topographs. This shows that dislocations observed in common inorganic crystals and organic crystals of small molecules can also be introduced even into protein crystals. The shape and configuration of dislocations strongly depended on the crystal form. This suggests that the growth mechanisms in the different crystal forms studied may differ. X-ray topography provides a useful tool for the characterization of protein crystal dislocations.
1. Introduction A detailed understanding of the function of proteins is facilitated by the knowledge of their three-dimensional structure. Despite enormous progress over the past decade in the structure analysis of proteins by X-ray and neutron crystallography, it is still largely limited by the difficulty of obtaining high-quality crystals.1,2 Protein crystals include a variety of crystal defects that limit the accuracy of structure analysis. An important objective for fundamental studies of protein crystal growth is to identify and reduce these crystal defects.3,4 X-ray topography is one of the most powerful methods for the characterization of crystal defects, especially dislocations. Since 1996, many groups have studied protein crystals with X-ray topography.5–16 However, the topographic contrasts from protein crystals are poor compared to those seen for common inorganic crystals and organic crystals of small molecules.17,18 The identification of the causes of topographic contrasts in protein crystals is still difficult. For successful X-ray topography of protein crystals, clear images showing the crystal defects need to be obtained. In this paper, we report this with the observation of clear images of crystal defects, especially dislocations, in protein crystals by synchrotron monochromatic-beam X-ray topography. Hen egg-white lysozyme (HEWL) has been extensively studied in terms of protein crystal growth and defects. It was selected for this study. HEWL is known to have multiple polymorphisms, for example, tetragonal, orthorhombic, monoclinic forms, etc., depending on variables such as the precipitant salts, pH, and temperature,19 which exhibit different morphologies as shown in Figure 1. The characterization of crystal defects in the multiple polymorphisms is important for the comprehensive understanding of protein crystal growth and intermolecular interaction. However, almost all studies on * Corresponding author. Tel: 81-45-787-2307. Fax: 81-45-787-2307. E-mail:
[email protected]. † Part of the special issue on (Vol 7, issue 11) the 11th International Conference on the Crystallization of Biological Macromolecules, Québec, Canada, August 16–21, 2006 (preconference August 13–16, 2006).
HEWL crystals have been carried out for only the tetragonal form. Thus, we show here X-ray topographs of not only the tetragonal form but also the orthorhombic and monoclinic forms.
2. Experimental Section 2.1. Crystal Growth. Six times crystallized HEWL (Seikagaku Kogyo Co. Ltd.) was used without further purification. All other chemicals used for preparing solutions were of reagent grade. Tetragonal HEWL crystals were grown by a salt concentration gradient method suggested by Ataka and Katsura (1992).20 A 5% HEWL solution was made in distilled water. The solution was adjusted to pH 4.4 with HCl. A precipitant NiCl2 was put on the bottom of the test tube held vertically, to which the lysozyme solution was slowly added. The test tube was sealed with parafilm and kept in a clean room at 23 °C. The NiCl2 naturally diffuses upward so that the concentration gradient, that is, solubility distribution, is formed along the tube. The crystallization mainly occurs at the minimum point of solubility after 1–2 days. Note that the solubility is a minimum at ∼8% NiCl2. Thus, the number of nuclei is limited so that large crystals are grown. After two weeks, large crystals up to ∼4 mm were obtained in the solution.21,22 The crystals had a tetragonal structure with space group P43212, lattice constants of a ) b ) 79.1 Å, c ) 37.9 Å, and eight molecules per unit cell. The grown crystals were bounded by the habit crystallographic faces of {110} and {101}. Orthorhombic HEWL crystals were grown by a liquid–liquid interfacial precipitation method suggested by Adachi et al. (2003).23 A HEWL solution containing 53 mg/mL HEW lysozyme and 3.5% NaCl at pH 4.7 was prepared. The HEWL solution was carefully added onto Fluorinert liquid with a high density of 1940 kg/m3 that was previously poured into a bottle. The bottle containing the interface of the HEWL solution and the Fluorinert liquid was placed in a thermostatic bath and maintained at 40 ( 0.1 °C. After approximately two weeks, large crystals up to 20 mm were grown at the interface. The crystals were orthorhombic with space group P212121, lattice constants of a ) 56.4 Å, b ) 73.7 Å, c ) 30.4 Å, and four molecules per unit cell.24 The crystals were bounded by the habit crystallographic faces of {110}, {010}, and {011}. Monoclinic HEWL crystals were also grown by the liquid–liquid interfacial precipitation method. A HEWL solution containing 5 mg/ mL HEWL and 2.5% NaNO3 at pH 4.5 was used. The crystal growth was carried out at 23 ( 0.1 °C. After about two weeks, large crystals up to 2 mm were grown. The crystals were monoclinic with space group P21, lattice constants of a ) 28.0 Å, b ) 62.5 Å, c ) 60.9 Å, R ) γ
10.1021/cg7009447 CCC: $37.00 2007 American Chemical Society Published on Web 11/07/2007
Synchrotron X-ray Topography of Protein Crystals
Crystal Growth & Design, Vol. 7, No. 11, 2007 2183 was mounted on the goniometer. A habit crystallographic face of the crystal was adjusted to be almost normal to the incident beam. An X-ray flat panel sensor (C9732DK, Hamamatsu Photonics KK) was employed to find reflections of interest for X-ray topography. The exact Bragg angle for a topographic reflection was determined by the measurement of the peak intensity of the reflection spot found on the flat panel sensor. X-ray film or nuclear plate was then used. The camera length was 25 cm. The monochromatic-beam topographs were recorded on X-ray films (Agfa D2) and nuclear plates (Ilford L4) with exposure times of about 10 s and 10 min, respectively.
3. Results and Discussion The most common contrast of crystal defects in X-ray topographs is the kinematical one (so-called direct image), produced by an additional diffracted intensity coming from distorted lattice areas close to each defect.18,26–28 The direct image can be obtained when µt < 1, where µ is the linear absorption coefficient and t is the crystal thickness. This condition determines the upper limit in crystal thickness. The further condition is that t > 0.4ξg, where ξg is the extinction length.29 This exhibits the lower limit in crystal thickness. Thus, the crystal thickness for the direct image is limited by the following condition: 0.4ξg < t < 1/µ
(1)
The extinction length ξg for a symmetrical reflection in a perfect crystal27,28 is given by the following equation: ξg ) (π/re)Vc cos θB/FhklλC
Figure 1. Schematic figures of morphologies of (a) tetragonal, (b) orthorhombic, and (c) monoclinic HEWL crystals. ) 90°, β ) 90.8°, and four molecules per unit cell.25 The crystals were j and {10 j 1}. j bounded by the habit crystallographic faces of {010}, {101}, Large crystals of more than 1.5 mm in thickness were selected for X-ray topographic experiments. 2.2. X-ray Topography. X-ray topography was carried out with synchrotron radiation in BL15B1 and BL14B at the Photon Factory (PF) of the High Energy Accelerator Research Organization (KEK) and in BL28B2 at SPring-8 with a monochromatic-beam of 0.4 or 1.2 Å. The synchrotron radiation was strongly scattered in the test tube or glass bottle in which HEWL crystals were grown. For synchrotron X-ray topography, a crystal in the test tube or glass bottle was gently transferred to a thin container, for example, a short straw, which is transparent for the synchrotron radiation. To avoid the evaporation of the water contained in the crystal, it was surrounded in the growth solution, and both sides of the straw were sealed with parafilm. The sealed straw
(2)
where re is the classical electron radius (re ) 2.82 × 10-5 Å), Fhkl is the structure factor, Vc is the volume of unit cell, C is the polarization factor (C ) 1 in our experiments), λ is the wavelength of X-ray radiation, and θB is the Bragg angle. It is clear from eq 2 that the extinction lengths for protein crystals can be far larger than those for most organic crystals of small molecules because of the extremely weak X-ray scattering power (Fhkl/Vc) of the former. Roughly speaking, the extinction length for a typical reflection at 1 Å radiation varies from millimeters to tens of millimeters for protein crystals, compared with a few micrometers to several tens of micrometers in most organic crystals of small molecules. For example, the extinction length is approximately 1.6 mm even for 440, a strong reflection of tetragonal HEWL crystals. The large extinction length can make it difficult to obtain the direct image of defects in protein crystals. If the crystal thickness is less than 0.4ξg, the defect contrast normally disappears. For real protein crystals, typically less than 0.5 mm in size, the defect contrast would probably be either weak or absent in X-ray topographs. To visualize defects, especially dislocations, in terms of direct images, one needs to use the strongest reflections, together with the use of extremely large crystals whose thickness is comparable to the extinction lengths of the reflections. However, such a strategy has been hardly carried out because of the difficulty of obtaining large crystals of more than 1 mm. Only our group has reported on white-beam X-ray topography with large protein crystals exceeding 1 mm.9,30,31 As a result, relatively clear images of dislocations were observed on Laue topographs. Therefore, it is expected that clearer images, similar to those that have been observed for common inorganic crystals and organic crystals of small molecules, are obtained by means of monochromatic-beam X-ray topography with protein crystals of more than 1 mm. The width of a direct image of a dislocation on X-ray topographs is proportional to ξg and g · b, where g is the diffraction vector (reflection) for the topograph and b is the
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Figure 3. X-ray topograph of an orthorhombic HEWL crystal in 0010 reflection taken with the incident beam almost normal to the (110) crystallographic face. OS1 and OS2 are the straight dislocations, OL is the dislocation loop, and OSB is the growth sector boundary.
Figure 2. (a) X-ray topograph and (b) its expanded figure of a tetragonal j reflection taken with the incident beam almost HEWL crystal in 4j40 normal to the (001) crystallographic face. TS1 and TS2 are the straight dislocations, TC is the curved dislocation, and TF is the fringe.
Burgers vector of the dislocation.32 For protein crystals with large extinction lengths and Burgers vectors, the image width of dislocations would be very large.33 The image width is a maximum at the exact Bragg angle, that is, at the top of the rocking curve, for the topographic reflection. Consequently, individual dislocation images can overlap, and the dislocations cannot be seen individually, especially for protein crystals. However, the image width decreases and narrows when the crystal is set far from the exact Bragg angle, that is, at the high or low angle flank of the rocking curve, where the average diffraction intensity from the crystal decreases. This is the socalled weak-beam technique. Thus, the weak-beam technique would be useful for the observation of individual dislocations in protein crystals. Figures 2–4 show monochromatic-beam X-ray topographs in j 0010, and 020 reflections, taken with the incident beam 4j40, j of tetragonal, orthoralmost normal to (001), (110), and (101) hombic, and monoclinic forms, respectively. For these topographic reflections, the values of 0.4ξg are 0.5, 1.6 and 0.2 mm, respectively, as shown in Table 1. All these values are less than the crystal size of more than 1.5 mm used in the experiments. Therefore, the crystal size in this study satisfies the critical condition for direct image as indicated by eq 1. In addition, the topographs were taken under the weak-beam condition away
Figure 4. (a) X-ray topograph and (b) its expanded figure of a monoclinic HEWL crystal in 020 reflection taken with the incident beam almost normal to the (110) crystallographic face. MC is the curved dislocation, and MF1 and MF2 are the fringe.
from the exact Bragg condition by about 0.005°. As a result, extremely clear images were obtained as shown in Figures 2–4. Note that the line images correspond to dislocation lines. These images are much clearer compared to those for any protein crystals that have been reported previously.5–16 As seen in Figures 3 and 4, large regions of black and white (clear) indicated by B and W, respectively, are observed as
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Table 1. The Minimum Criterion of 0.4 ξg in Crystal Thickness for j 0010, and 020 reflections of Tetragonal, Direct Images in 4j40, Orthorhombic, And Monoclinic HEWL Crystalsa crystal form
reflection j tetragonal 4j40 orthorhombic 0010 monoclinic 020
Vc (×10-16 θB λ (deg) (Å) mm3) 2.37 1.27 1.07
2.46 3.77 1.09
Fhkl
1.2 1669 0.4 907 1.2 1660
ξg 0.4ξg (mm) (mm) 1.3 3.9 0.6
0.5 1.6 0.2
a Note that λ is the wavelength of X-ray radiation, Fhkl is the structure factor, ξg is the extinction length, Vc is the volume of unit cell, and θB is the Bragg angle.
background in the crystal image. Such contrasts have been often observed for larger crystal samples. The black and white (clear) regions correspond to ones that satisfy and are far from the exact Bragg condition, respectively. The black part shifted toward the original white (clear) one by rotating the crystal sample, so that the black and white (clear) parts changed to opposite contrasts. The shift of the black part implies that part to fulfill the exact Bragg condition by rotating the sample. This suggests that the reflection planes in the black part are slightly tilted to those in the white (clear) one. Therefore, the crystal sample is slightly distorted all over the crystal. The distortion might be introduced during the crystal growth or by handling after the growth. This can be related to soft and fragile characteristics of protein crystals.21,34,35 For the tetragonal form, as shown in Figure 2, not only straight dislocations (marked TS1 and TS2) but also curved dislocations (marked TC) are clearly observed on the topograph. The straight dislocations originate at or around the central nucleus and run outward to the crystal surfaces. They run approximately along the direction with the {110} growing face. Such a configuration of the straight dislocations is similar to that of screw dislocations with the Burgers vector identified by white-beam topography previously.16,30 Thus, it is likely that the straight dislocations TS1 observed in this study are grown-in screw dislocations with the [110] Burgers vector. In addition, it should be noted that the straight dislocations TS1 are clearly observed as a pair as indicated by arrows. This pair feature is a typical example for dislocations arising during solution growth.36 On the other hand, the other pair dislocations (marked TS2) j j almost parallel to the [110] direction are observed. If the [110] j dislocations are of screw character with the [110] Burgers vector, j reflection. However, their their images disappeared in the 4j40 images clearly appeared on the topograph as shown in Figure j 2a. This means that the [110] dislocations are not of screw j character with the [110] Burgers vector. The character of dislocations TS2 might be different from the screw one of TS1 despite their similar shape and configuration. Thus, it is concluded that there are at least two kinds of pair straight dislocations with different characters almost parallel to in tetragonal form. Moreover, as shown in Figure 2b, some fringes marked TF are observed. Similar fringes have been observed for previous X-ray topographs taken with the incident beam almost normal to (110) in tetragonal form.10 These fringes appear at the boundaries between different growth sectors. The different growth sectors are misaligned at the boundaries. Consequently, the misaligned growth sectors could produce some degree of stress inside the crystal lattice and wedge-shaped volumes. Therefore, the origin of the observed fringes is considered to be due to Moire fringes due to the superposition in the direction of the diffracted beam of different crystal volumes having
slightly different misalignment or lattice parameters37 or Pendellösung fringes due to the presence of wedge-shaped crystal volumes.38 For the orthorhombic form, as shown in Figure 3, not only dislocation lines but also growth sector boundaries (marked OSB) were clearly observed. The majority of dislocations (marked OS1 and OS2) are the straight ones and seem to emerge j on the (110) surface. From their running directions, they can be classified into two types of dislocations. One is the bended dislocation (marked OS1) that originates at or around the central nucleus. It first runs along the growth sector boundary and then j bends at a site and runs along the [110] direction. The other is the simple straight dislocation that nucleates at or around the j growth sector boundary and runs along the [110] direction. The latter straight dislocation is also the pair type one, similar to those observed in tetragonal form as mentioned above that is j typical for solution growth. The [110] dislocation is similar to those of the edge character with the [001] Burgers vector that has been identified in our recent study with white-beam X-ray topography.39 In addition, small loop dislocations (marked OL) were clearly observed, although the origin is still unclear. For the monoclinic form, as shown in Figure 4a, the shape and configuration of dislocation lines are quite different from those of tetragonal and orthorhombic forms as mentioned above. Almost all dislocations exhibited loop and largely curved ones (marked MC). Some fringes (marked MF1) were also observed. Such a difference can be related to the growth mechanism and the intermolecular interaction. In addition, some of curved dislocations marked MC look like a sequence of loops that originated from a source. This lets us imagine the multiplication and moving of dislocations, although detailed analysis is not carried out yet. As shown in Figure 4b, wedge-like regions with fringes (marked MF2) are also observed. The wedge shape corresponds j surface of the to a hollow opening that developed on the (101) crystal. The hollow opening is considered to be due to the Berg effect, resulting in an enhanced growth rate at the crystal edge.25 For the monoclinic form, the Berg effect provides the anisotropic one for the crystal growth. Namely, the growth rate of the +b face is much faster than that of the -b face. Consequently, the shapes of (010) faces appear differently on +b and -b sides, and/or the hollow opening exhibits a wedge-like shape. This hollow opening was clearly observed with a fringe pattern. The analysis of the fringe might lead to the detailed understanding of growth mechanism and the Berg effect. The anisotropic property due to the Berg effect seems not to influence the shape and configuration of the dislocations as shown in Figure 4b.
4. Conclusion We have shown well-defined topographic images in tetragonal, orthorhombic, and monoclinic HEWL crystals, compared with those have been observed in any protein crystals so far.5–16 It was found that various types (straight, curve, loop, and pair) of dislocations that have been observed in common inorganic crystals and organic crystals of small molecules are also seen in protein crystals. In addition, the multiplication and moving of dislocations in protein crystals is expected from the shape and configuration of curved dislocations seen in the monoclinic form. Detailed topographic images, such as those in this paper, will lead to a more detailed understanding of crystal growth and defects in protein crystals. Acknowledgment. We thank Dr. H. Sugiyama and Dr. K. Hirano of KEK and Dr. K. Kajiwara of the Japan Synchrotron
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Radiation Research Institute (JASRI) for their help on synchrotron radiation X-ray topography. The synchrotron radiation X-ray topography was performed at PF under the auspices of the Photon Factory Program Advisory Committee of KEK (Proposal No. 2005G015, 2006G260) and at the SPring-8 with the approval of JASRI (Proposal No. 2005A0405-ND3c-np). M.T. and K.K. were supported by the Grant-in-Aid for Science Research (No. 18560648) from the Ministry of Education, Culture, Sports, Science and Technology. Supporting Information Available: Some details in the theoretical background of X-ray topography and contrast formation. This material is available free of charge via the Internet at http://pubs.acs.org.
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