DOI: 10.1021/cg100790r
Single-Crystal X-ray Diffraction Study of a Magnetically Oriented Microcrystal Array of Lysozyme
2011, Vol. 11 12–15
Fumiko Kimura,† Kimihiko Mizutani,‡ Bunzo Mikami,‡ and Tsunehisa Kimura*,† †
Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan, and ‡Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Received June 13, 2010; Revised Manuscript Received October 24, 2010
ABSTRACT: Single-crystal X-ray diffraction analysis was performed on a magnetically oriented microcrystal array (MOMA; a composite in which microcrystals are oriented three-dimensionally in a polymer matrix) to demonstrate its potential to determine protein structure from a powder sample. Recrystallized hen egg-white lysozyme was pulverized to prepare a microcrystalline powder as a model system from which a MOMA was prepared. The microcrystalline powder was suspended in an ultraviolet (UV)-curable monomer and rotated nonuniformly in a static magnetic field (8 T), and then, the crystalline alignment was consolidated by UV light irradiation. The obtained sample was subjected to conventional single-crystal X-ray diffraction measurement. The structure determined for the MOMA was found to belong to the space group P212121 with unit-cell dimensions a = 51.26, b = 59.79, and c = 29.95 A˚, in agreement with the structure of the single crystal from which the MOMA was prepared. The protein structure was refined at the highest resolution of 3.0 A˚, which agreed with that determined with an independently grown single crystal under comparable conditions. The fabrication method presented here provides a powerful means of determining the structure of proteins that do not crystallize to sizes suitable for conventional single-crystal X-ray analyses. The three-dimensional structure of biomolecules is of great importance because it is closely related to biological functions. A protein expresses its functions when binding with ligands; a drug molecule functions by binding to a specific protein site.1,2 Therefore, the structure determination of proteins is a key issue in the development of new drug molecules. There are three major methods to solve the structure of proteins: solution nuclear magnetic resonance (NMR),3 X-ray singlecrystal,4-6 and X-ray powder diffraction7,8 methods. The solution NMR method has an advantage over diffraction methods in that it does not require crystals, but it can be applied only to proteins with lower molecular weights. X-ray single-crystal analysis is the most powerful method, but it is sometimes difficult to grow a crystal9 sufficiently large for conventional or synchrotron single-crystal X-ray measurement. Note that the size requirement is much more important for neutron diffraction measurement.10 On the other hand, the X-ray powder method is employed if microcrystalline powders are available, but an appropriate refinement of many parameters is needed for a successful result. We recently proposed a fabrication method for a magnetically oriented microcrystal array (MOMA) (in previous papers11-15 we called this array a pseudo-single crystal (PSC); hereafter, we will use the term MOMA). A MOMA is a composite in which microcrystals are aligned three-dimensionally. Microcrystals are suspended in an ultraviolet (UV)-curable monomer and rotated nonuniformly in a static magnetic field to achieve threedimensional crystal alignment. Then, the monomer is photopolymerized to maintain the achieved alignment. The fabrication method of MOMA is applicable to biaxial crystal systems such as triclinic, monoclinic, and orthorhombic systems. The magnetic susceptibility tensor of these crystal systems possesses three different principal values, which is necessary for achieving the three-dimensional magnetic alignment. For this reason, the orthorhombic form of lysozyme has been chosen in the present study. We have so far demonstrated that the X-ray diffraction data from the MOMA is equivalent to that of a corresponding single crystal. We fabricated MOMAs from inorganic13 and organic14 microcrystalline powders and successfully performed crystallographic analyses of the microcrystals from which the *Corresponding author: E-mail:
[email protected]. pubs.acs.org/crystal
Published on Web 11/17/2010
MOMAs were prepared. The structures determined13,14 using MOMA were in good agreement with those reported in the literature where single crystal samples were used. It was also demonstrated that this method is more advantageous when applied to single-crystal neutron diffraction analysis because a MOMA can be fabricated to centimeter sizes.15 Although a number of studies have reported on magnetic effects on the crystal orientation and growth of proteins,16-20 no attempt has been made so far to apply the MOMA method to protein structure analysis. In this paper, we report a single-crystal X-ray analysis of a MOMA prepared from a lysozyme microcrystalline powder to demonstrate that the MOMA method is also useful in the structure determination of biomacromolecules. An as-received sample (hen egg-white lysozyme, Maruwa Food Industries, Inc.) was recrystallized via a batch method at 313 K. This temperature was selected in order to obtain an orthorhombic form.20 We dissolved 2 g of lysozyme in 5 mL of 50 mM acetate buffer (pH 4.5), and 1.67 mL of acetate buffer containing NaCl (concentration: 50 mg/mL) was then added, followed by the addition of 3.33 mL of a solution of 50 wt/wt % polyethylene glycol (MW = 20 000) in acetate buffer. The container holding the prepared solution was kept in a water bath at 313 K for 3 days. The crystals were separated by filtration, dried in air, and stored at 277 K. Using a mortar, we pulverized 20 mg of recrystallized lysozyme, which was then dispersed in 90 mg of UV-curable monomer (No. 801SE-LHF of Kyoritsu Chemical and Co., Ltd.; viscosity, 2.0 Pa s). The suspension thus obtained was allowed to stand for a few days. Then, the suspension was poured into a plastic container (diameter, 5 mm; height, ca. 8 mm). The drying process is necessary because it is difficult to homogeneously suspend the microcrystals in a UV-curable monomer if they are wet. However, the drying process is not essential: if microcrystals are used in the form of an aqueous suspension and there is an appropriate water-soluble UV-curable monomer or gelating matrix that does not dissolve or coagulate the microcrystals and is capable of consolidating the alignment, then the current method is applicable to any proteins. The container was mounted on a sample-rotating unit placed at the bore center of a cryogen-free superconducting magnet (Sumitomo Heavy Industry), generating a horizontal magnetic field of 8 T. The rotation axis was vertical. The rotation was not r 2010 American Chemical Society
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Figure 1. Nonuniform rotation of sample in a static horizontal magnetic field B. The rotation speed is switched between ωs and ωq every time the x0 axis passes the switching boundaries, where ωs < ωq and 0° χ2 > χ3. The parameters R, ωs, and ωq were tuned so as to minimize and equalize the half-widths of the diffraction spots. Broadening of the half-width occurs through thermal fluctuations of the χ1 and χ3 axes about the x0 and z0 coordinates, respectively. An inappropriate choice of these parameters increases the half-width; for example, if ωs =ωq, the χ1 axis is not fixed to a specific direction, resulting in a broadening of spots or in a ring pattern. The suspension was rotated in a static field of 8 T for about 50 min, and the achieved alignment was consolidated by UV-light irradiation for 15 min in the magnetic field to obtain a MOMA. Nonuniform rotation was essential to achieve the three-dimensional alignment of magnetic axes.11,12 A specimen of 1.5 1.5 3.0 mm3 was cut from the obtained MOMA. Figure 2a shows the MOMA sample used for X-ray measurement. The microphotograph is shown in Figure 2b; the size of microcrystals was ca. 5-50 μm. The minimum size of a microcrystal required for magnetic alignment is determined by the relationship between its magnetic energy and thermal energy.11 Because the magnetic susceptibility values of lysozyme are not available, the minimum size cannot be estimated accurately. However, supposing that the magnetic nature of a lysozyme crystal is similar to that of organic crystals, it seems that a 5 μm microcrystal is fairly large to undergo significant alignment under 8 T. The specimen was mounted on a wire, and the X-ray experiments were carried out on an MAC Science Dip 2000 diffractometer equipped with an MXP18HF22 rotating anode generator (45 kV, 84 mA). Graphite-monochromated Cu KR radiation was used. The collimator size was 0.9 mm. Data was collected via ω scans from 45.0° to 244.0° at 1.0° steps and to a maximum 2θ of 33.7° at room temperature. The crystal-todetector distance was 150.0 mm. A total of 200 oscillation images were collected. The divergence of the X-ray beam was 0.5°. Data was processed with IPMOSFLM software. Refined atomic coordinates (PDB code 1VDQ) were used for the starting model for structural refinement. The MOLREP program from the CCP4 software suite was used for molecular replacement, followed by refinement of the atomic positions and isotropic thermal factors using phenix.refine software. Further modeling was performed using coot software, and a second refinement was carried out using phenix.refine software at the highest resolution of 3.00 A˚. The fluctuations of the χ1 and χ3 axes were estimated from the half-widths determined from the rocking curve and the azimuthal β scan, respectively. The rotation axis of the ω scan coincided with the χ3 axis. The data was collected using ω scans at 0.5° steps.
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Figure 2. (a) Photograph (a division: 1 mm) of a magnetically oriented microcrystal array (MOMA) of lysozyme. (b) Microphotograph of MOMA that is composed of various sizes of microcrystals. Table 1. Fluctuations of χ1 and χ3 for Three Sets of Parameters (R, ωs, ωq) Determined Using the (400) Diffraction Spota R (deg)
ωq (rpm)
ωs (rpm)
fluctuation of χ1 (deg)
fluctuation of χ3 (deg)
10 10 20
100 60 60
30 10 10
5.7 4.5 6
NA 4.3 5.1
a The fluctuation of χ1 is determined by the half-width of the rocking curve, and the fluctuation of χ3 is determined from the azimuthal β scan.
The three parameters, ωs, ωq, and R, must be selected appropriately in order to obtain sharp diffraction spots. The half-width of a spot is a result of the fluctuations of the χ1 and χ3 axes about the x0 and z0 axes, respectively. In terms of diffraction analysis, it is advantageous that the magnitudes of the two fluctuations are equal. From previous studies,11,12 it is known that the slow rotation speed ωs should satisfy ωsτ . 1/2 (Rapid Rotation Regime (RRR)) for sharp diffraction spots to be obtained; here, τ is the time constant of the alignment of the easy-magnetization axis χ1 under a static magnetic field and is expressed by τ ¼ 6μ0 η=χa B2
ð1Þ
where μ0 is the magnetic permeability of vacuum, B is the magnetic flux density, χa is the anisotropic magnetic susceptibility, and η is the viscosity of the medium. Here, it is assumed that a crystallite is magnetically uniaxial and that its shape is spherical. Using χa =10-8, B = 8 T, and η = 2.0 Pa s as an approximation, we obtain τ = 24 s. The value of ωs = 10 rpm (=1 rad/s) used in this study then satisfies the condition ωsτ . 1/2. Next, an appropriate choice of R and ωq is necessary in order to minimize and equalize the fluctuations of the χ1 and χ3 axes. In previous studies13,14 describing the single-crystal analysis of MOMAs, R=90° was appropriate. However, a theoretical study21 shows that the equalization cannot be achieved for some sets of ( χ1, χ2, χ3) if R is fixed to 90°. Since the three values of the magnetic susceptibility of lysozyme are unknown, we need to find out an appropriate value of R by trial and error. Table 1 summarizes the half-widths of a diffraction spot (the (400) diffraction) obtained from MOMAs fabricated with some sets of parameters (ωs, ωq, R). We tested several combinations of the parameters, and R = 10°, ωs =10 rpm, and ωq =60 rpm was found to be the most suitable regarding minimization and equalization of the half-widths. This condition was therefore chosen for the preparation of a MOMA for X-ray analysis. In Figure 3, diffraction patterns taken from three directions are shown. Well-separated diffraction spots were obtained. The directions of the magnetic axes as deduced from the preparation procedure are indicated in the figure. From the results of the indexing described in the next paragraph, the crystal belongs to the orthorhombic system. For this crystal system, the magnetic axes correspond to the crystallographic axes. From the figure, we find that the cell dimensions increase in the order χ1, χ2, and χ3.
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Kimura et al.
)
)
)
Figure 3. X-ray diffraction images of a magnetically oriented microcrystal array (MOMA) obtained through the ω scan for 0-45°. The MOMA was prepared under the conditions of R = 10°, ωs = 10 rpm, and ωq = 60 rpm. The directions of magnetic axes are indicated in the figure. Here, χ1 c, χ2 a, and χ3 b. Table 2. Summary of Data Collection and Refinementa A. Crystal Data wavelength (A˚) space group cell dimensions a (A˚) b (A˚) c (A˚) V (A˚3) observed reflections resolution (A˚) independent reflections completeness (%) Rsym (%) redundancy mean I/σ
1.5418 P212121 51.26 59.79 29.95 91,803 10,604 26.77-3.00 (3.16-3.00) 1828 (250) 89.6 (88.6) 19.4 (48.7) 5.8 (5.5) 9.3 (2.7) B. Refinement Statistics
Figure 4. X-ray diffraction image with resolution rings indicated.
)
)
)
Combining the indexing results, we conclude that χ1 c, χ2 a, and χ3 b. The alignment of the c axis parallel to the magnetic field has been reported for orthorhombic lysozyme crystals.20 Figure 4 shows a detailed diffraction pattern with resolution rings. The X-ray results are summarized in Table 2. The indexing was determined as follows: space group, P212121; lattice constants, a = 51.26 A˚, b = 59.79 A˚, c = 29.95 A˚, and V = 91 803 A˚3. This data was compared with that reported on the lysozyme single crystal (PDB code 2ZQ4) in Table 3. The cell dimensions obtained in this study are shorter than those reported in the literature. The shrinkage was attributed to the dehydration of the crystal that was inevitable in the present study, as described earlier. X-ray diffraction of dehydrated lysozyme crystals in triclinic22 and monoclinic23 forms has been reported. The graphical display is shown in Figure 5 for easy comparison of the MOMA with the reported structure (PDB code 2ZQ4). A comparison of the CR positions between the present result and 2ZQ4 gave rmsd = 0.755 A˚, indicating that the shrinkage of the lattice was mainly due to the loss of water molecules and that the protein chain conformation remained essentially unchanged. In conclusion, we have demonstrated that single-crystal X-ray diffraction analysis is possible for a protein microcrystalline powder by using our MOMA method. In this study, a lysozyme microcrystalline powder was used for demonstration purposes. The microcrystalline powder, suspended in a UV-curable monomer, was aligned three-dimensionally under modulated sample rotation in a static magnetic field, and the alignment achieved was
resolution limits (A˚) no. of reflections used completeness (%) no. of protein atoms no. of solvent molecules final R-factor free R value average B-factor (A˚2) rms deviation from ideal geometry bond distances (A˚) bond angles (deg) dihedrals (deg) a
25.86-3.00 (3.78-3.00) 2989 (1423) 84.4 (81) 1001 0 0.215 (0.253) 0.270 (0.292) 51.0 0.004 0.743 22.596
The values in the highest resolution bin are indicated in parentheses.
Table 3. Comparison of Crystal Data sample
MOMA
single crystal
PDB code space group cell dimensions a (A˚) b (A˚) c (A˚) V (A˚3)
present work P212121
2ZQ4 P212121
51.26 59.79 29.95 91,803
56.48 73.76 30.54 127,229
consolidated by UV-light irradiation. The MOMA was successfully indexed, and its structure was solved using the standard method of protein single-crystal analysis. The resolution was not very high because the dehydration was inevitable in the present study. One possible way to improve the resolution is to use an UV-curable monomer that disperses hydrated microcrystals. Alternatively, gelation of an aligned aqueous suspension of microcrystals might work to improve the resolution. The method presented here provides a powerful means of performing the
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Figure 5. Comparison of the crystal structures of lysozyme:24 (a and b) the structure determined through a MOMA prepared in the present study; (c and d) the structure reproduced from the database (PDB code 2ZQ4).
single-crystal X-ray diffraction analysis of proteins that do not crystallize to a size necessary for conventional X-ray measurement. Acknowledgment. This work was partially supported by the JSPS Asian Core Program “Construction of the World Center on Electromagnetic Processing of Materials”. This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B), 20350106, 2008.
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