Growth of Large Protein Crystals by Top-Seeded Solution Growth

Nov 3, 2009 - Together with the Floating and Solution-Stirring Technique. Noriko Shimizu,† Shigeru Sugiyama,†,⊥ Mihoko Maruyama,†,⊥ Hiroshi ...
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DOI: 10.1021/cg900740n

Growth of Large Protein Crystals by Top-Seeded Solution Growth Together with the Floating and Solution-Stirring Technique

2009, Vol. 9 5227–5232

Noriko Shimizu,† Shigeru Sugiyama,†,^ Mihoko Maruyama,†,^ Hiroshi Y. Yoshikawa,†,^ Yoshinori Takahashi,†,^ Hiroaki Adachi,†,‡,^ Kazufumi Takano,†,‡,^ Satoshi Murakami,‡,§,^ Tsuyoshi Inoue,†,‡,^ Hiroyoshi Matsumura,*,†,‡,^ and Yusuke Mori†,‡,^ †

Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan, ‡SOSHO Inc., Osaka 541-0053, Japan, §Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan, and ^CREST JST, Suita, Osaka 565-0871, Japan Received July 2, 2009; Revised Manuscript Received September 30, 2009

ABSTRACT: Neutron protein crystallography is expected to yield a more precise understanding of the protein structure by visualizing hydrogen atoms and hydration water molecules. However, relatively few protein structures have been analyzed by neutron diffraction, in part because very large crystals of the target proteins are required in order to compensate for the weak flux of the available neutron beam. Here, we report the crystal growth of hen egg white lysozyme (HEWL) by the top-seeded solution growth with the floating and solution-stirring method (TSSG-FAST), the first utilized for protein crystallization. A series of crystal-growth experiments demonstrated that TSSG-FAST is an efficient strategy for rapidly obtaining large single crystals and effectively preventing polycrystallization of the seed crystal. Furthermore, the shape of the HEWL crystals was found to be strongly influenced by the orientation of the seed crystal, suggesting that the shape of the HEWL crystal may be controlled by TSSG-FAST. It is anticipated that this new approach will expand the limits on the growth of large protein crystals for neutron protein crystallography.

*To whom correspondence should be addressed. E-mail: matsumura@ chem.eng.osaka-u.ac.jp.

grow at the interface of the two liquids, thereby preventing protein crystals from adhering to the growth vessel. As a result, the crystals can be handled without causing mechanical damage to them, in the same manner as in containerless techniques.13,14 Indeed, this method was applied to a repeated macroseeding procedure to grow large crystals (approximately 1.9  1.8  0.4 mm in size) of HIV protease-inhibitor complex.9 In order to shorten the time required for crystal growth (the second disadvantage), we further developed the floating and stirring technique (FAST).10,15,16 This technique is essentially the same as the two-liquid method, except that the solution is stirred by a shaker or magnetic stirrer. The growth rate of the hen egg white lysozyme (HEWL) crystals was found to be accelerated by the stirring.1,15,16 However, despite these improvements, little effort has been made to overcome the other disadvantages (i.e., suppression of polycrystallization and control of the crystal shape). Top-seeded solution growth (TSSG) has been widely utilized for growing inorganic-compound crystals to prevent polycrystallization and to produce high-quality large single crystals.17,18 In TSSG, a seed crystal is hung by a seed crystal holder from the top of the growth vessel. We have also previously reported a modified technique for growing CsLiB6O10 crystals. In the course of these studies,18-20 TSSG was combined with the solution-stirring method, further improving the subsequent growth of CsLiB6O10 crystals.18-20 Thus, TSSG is a powerful tool for obtaining high-quality and large inorganic-compound crystals; however, the method has never been applied for the growth of protein crystals, in part because hanging the fragile protein crystals is extremely difficult. Herein, we first applied TSSG to the growth of protein crystals. To hang the protein crystals, we applied the twoliquid system, in which protein crystals floats on Fluorinert. It was found that the floating crystals could be successfully

r 2009 American Chemical Society

Published on Web 11/03/2009

1. Introduction Neutron protein crystallography provides a powerful complement to X-ray analysis by enabling the visualization of hydrogen atoms and hydration water molecules,1,2 which play important roles in many biological and physiological functions. One significant objective in neutron protein crystallography is to grow large protein crystals (e.g., 1-10 mm3)3 in order to compensate for the weak flux of the available neutron beam.4,5 However, current techniques for large crystal growth in proteins have several inherent disadvantages. First, in the macroseeding technique that is frequently used to enlarge protein crystals, extreme care must be taken when the seed crystal is handled. This technique introduces a seed crystal into a pre-equilibrated protein solution, repeating this cycle several times. However, the resulting protein crystals are very soft and fragile,6,7 and if crystals adhere tightly to the growth vessel, it becomes increasingly difficult to handle them without causing mechanical damage. Second, a long growing time is often required for the growth of large protein crystals. For example, it has been reported that more than a month was required for large crystal growth in HIV protease-inhibitor complex.8,9 Third, crystals occasionally overlap each other and generate polycrystals during their growth, although a single crystal is essential for structural analysis. Fourth, protein crystals tend to grow in a long thin plate shape, even though large crystal volumes are required for neutron crystallography. To overcome the disadvantage of fragile crystals (the first described above), we previously developed a two-liquid batch method for growing high-quality protein crystals.10-12 In this method, a protein-precipitant solution floats on an insoluble and very dense liquid (Fluorinert FC-70) and protein crystals

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attached to a seed crystal holder with silicone glue, allowing us to apply the TSSG method to grow protein crystals. Comparative analyses of the growth of HEWL crystals demonstrated that the combination of TSSG with FAST (TSSGFAST) was a more efficient strategy for rapidly obtaining large single crystals and that TSSG-FAST reproducibly produced large single HEWL crystals without polycrystallization. In addition, the shape of the HEWL crystal was strongly influenced by the orientation of the seed crystal, indicating that the shape of the HEWL crystal may be controlled by TSSG-FAST. We suggest that this new approach will expand the limits on the growth of large protein crystals for neutron protein crystallography.

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Figure 1. Schematic illustration of the crystallization setup for TSSG-FAST.

2. Materials and Methods 2.1. Materials. HEWL was purchased from Seikagaku Corporation, Japan (catalogue No. 100940, recrystallized six times). The insoluble and very dense liquid (Fluorinert FC-70) was purchased from Sumitomo 3 M Limited, Japan. The glass growth vessel (26 mm diameter) was siliconized to prevent crystals from adhering to the vessel. The polyethylene seed crystal holder (29 mm long) was purchased from Sorenson TM BioScience, Inc., USA (catalogue No. 30471T). The vessel was covered by a cap made of polyethylene (30 mm outside diameter, 9 mm inside diameter). The clay by which the holder was fixed to the cap was purchased from DEBIKA Corporation, Japan (catalogue No. 090189). The silicone glue was purchased from GE Toshiba Silicones Co., Ltd., Japan (catalogue No. TSE387). 2.2. Crystallization. The HEWL solution was passed through 0.22 μm filters prior to crystallization. As seed crystals, the HEWL was crystallized at a protein concentration of 30 mg/mL using the batch method in 0.5 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5) at 293 K. A seed crystal was transferred into the interface between 4 mL of Fluorinert and 7 mL of a protein solution containing 20 mg/mL HEWL in 0.5 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5), which allowed subsequent growth of the crystals without causing mechanical damage. The degree of supersaturation was 1.2, which was calculated by the following equation: σ  ln (C/Ce), where C denotes the concentration of HEWL, and Ce denotes the solubility, respectively. The value of Ce was 6.22 mg/mL, according to the previous study.21 In the protein solution, a seed crystal was adhered to the seed holder with silicone glue and the seed holder was subsequently fixed to the cap by clay. After the glue solidified, the seed holder was lifted up from the interface with the seed crystal and was again fixed to the cap by clay. Thus, a seed crystal was lifted up from the interface between Fluorinert and a protein solution. The protein-precipitant solution was stirred by a rotary shaker or magnetic stirring. For stirring by a rotary shaker, the vessel was placed in the rotary shaker (60 rpm). For magnetic stirring, a magnetic stirrer bar was placed in the Fluorinert to mildly stir the floating protein-precipitant solution (100 rpm). All crystallization experiments were performed at 293 ( 1.0 K.

3. Results and Discussion We here report the first application of TSSG to the growth of protein crystals. First, we evaluated TSSG, FAST, and their combination (TSSG-FAST) for the growth of HEWL crystals. The comparative analysis demonstrated that the combined method TSSG-FAST was adequate for rapidly obtaining large single HEWL crystals. Furthermore, it appeared that the shape of the HEWL crystal was significantly affected by the orientation of the seed crystals, suggesting the new possibility of controlling the growth direction. The details of these experimental results are described below. 3.1. Setup of the TSSG-FAST Method for Growing HEWL Crystals. Figure 1 schematically illustrates the crystallization setup for TSSG-FAST. Fluorinert was used as the insoluble

and very dense liquid. The protein-precipitant solution was placed onto the Fluorinert to configure the two-liquid system. A key aspect of this technique is that the seed protein crystal is hung by a seed holder protruding from the top of the growth vessel. Initially, seed crystals of HEWL were obtained by the batch method as described in Materials and Methods. The seed crystals were then gently transferred to the proteinprecipitant solution on the Fluorinert in the growth vessel. A seed holder with silicone glue was carefully brought into contact with the seed crystal from the top of the vessel, and the seed holder was subsequently kept in contact with the seed crystal for 24 h to solidify the glue. To investigate the mechanical damage caused by the contact, X-ray measurement was performed using the adhered crystals, and it was confirmed that the crystal was single with smaller mosaicity (∼0.3 deg) (data not shown). Thus, this novel approach allowed a seed crystal to adhere to the holder without causing mechanical damage to it. It has been reported that various protein crystals can be captured without causing significant damage by Crystal Catcher, which directly captures a crystal with an adhesive.22,23 Therefore, our approach may also be applicable to various other protein crystals. The silicone glue used in this experiment has been utilized in previous studies of HEWL crystal growth, and it was proposed that the glue does not have serious impurity effects on crystal growth.24 We further obtained consistent results by a laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM).25,26 (The details will be described elsewhere). After adhesion of the seed crystal, the seed crystal was lifted 7 mm away from the two-liquid interface, and the protein-precipitant solution was stirred by rotary shaker (60 rpm) or by magnetic stirring (100 rpm) to grow the seed crystal. In a series of crystal growth experiments, HEWL crystals were grown at 293 ( 1.0 K. 3.2. Comparison of TSSG-FAST with TSSG. In this comparative analysis, HEWL crystals were grown by TSSG-FAST with a rotary shaker (60 rpm) and by TSSG without solution stirring (Figure 2). In TSSG-FAST, the growth vessel was put onto the rotary shaker (60 rpm), which allowed mild stirring of the crystallization solution.15 The crystallization condition, the amount of Fluorinert, and the growth vessels were exactly the same for TSSG-FAST and TSSG. Photographs of the setups are presented in Figure 2. The seed crystals had dimensions of 3.0  1.7  1.4 mm (TSSG-FAST) and 3.1  2.5  1.4 mm (TSSG). After seven days, the growth widths along the c axis of the HEWL crystals and the increases in the crystal volume were

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Figure 2. Photograph of the setups for TSSG-FAST (A) and TSSG (B). (A) Photographs of the setup for TSSG-FAST (stirred by rotary shaker at 60 rpm). The crystal was approximately 3.01.71.4 mm. The HEWL crystal grew from 3.0 mm to 3.6 mm along the c axis in 7 days. The crystal volume increased from 7.14 mm3 to 11.52 mm3. (B) Photograph of the setup for TSSG (without stirring). The crystal was approximately 3.1  2.5  1.4 mm. The HEWL crystal grew from 3.1 mm to 3.4 mm along the c axis in 7 days. The crystal volume increased from 10.85 mm3 to 11.97 mm3.

Figure 3. (A) Schematic illustration of tetragonal HEWL crystal. (B) Growth widths along the c axis of the HEWL crystals grown by TSSG-FAST and TSSG. With TSSG-FAST, the growth widths in 7 days along the c axis were 0.6 mm, and the growth rate was calculated to be 0.09 mm/day. In contrast, TSSG produced growth widths of 0.3 mm in 7 days, and the growth rate was calculated to be 0.04 mm/day. (C) Increases in crystal volume of the HEWL crystals grown by TSSG-FAST and TSSG. The increases in the crystal volume in 7 days were approximately 4.4 and 1.1 mm3 for TSSGFAST and TSSG, corresponding to growth rates of 0.63 and 0.16 mm3/day.

measured (Figure 3). In TSSG-FAST, the growth widths along the c axis were 0.6 mm, and the growth rates were calculated to be 0.09 mm/day. In contrast, TSSG had growth widths along the c axis of 0.3 mm, and their growth rates were calculated to be 0.04 mm/day. Thus, the growth rate along the c axis in TSSG-FAST was approximately twice as large as that in TSSG without solution stirring (Figure 3A,B). These results are consistent with a previous analysis of HEWL crystal growth by FAST and by the two-liquid system15 that indicated that the crystals grew roughly twice as fast with FAST than with the two-liquid system. Furthermore, the

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increases in the crystal volume were approximately 4.4 mm3 for TSSG-FAST and 1.1 mm3 for TSSG, corresponding to growth rates of 0.63 and 0.16 mm3/day (Figure 3C). To confirm the reproducibility, growth rates of the other seed crystals grown in same conditions were also measured (Figure S1, Supporting Information). These data also demonstrate that crystals grown by TSSG-FAST grew 1.4-2.1 times as fast as that by TSSG without solution stirring, although the promoted growth rates appeared to vary to some extent. We also confirmed that TSSG-FAST with magnetic stirring (100 rpm) produced similar increases in growth width and crystal volume (data not shown). Taken together, these results demonstrate that TSSG-FAST, rather than conventional TSSG, is likely to be an efficient strategy for promoting rapid growth of HEWL crystals. The question naturally arises as to why the solution stirring promotes the rapid growth of HEWL crystals. We observed by polyethylene beads that the flow velocity of TSSG-FAST was faster than that of TSSG. Therefore, the stirring solution must lead the increase in the protein concentration near the crystal surface. In addition, our LCMDIM observation demonstrated that the faster solution flow increased the number of 2D nucleation on the crystal surface (Maruyama et al., unpublished results). Thus, the growth rates were probably promoted by the increased number of 2D nucleation caused by stirring solution, thereby promoting the rapid growth of HEWL crystals. 3.3. Comparison of TSSG-FAST with FAST. To evaluate the effectiveness of TSSG, TSSG-FAST was compared with conventional FAST for the growth of HEWL crystals. In both TSSG-FAST and FAST, the HEWL crystals were grown with magnetic stirring (100 rpm). The crystallization conditions, the amount of Fluorinert, and the growth vessels were exactly the same in the comparative experiment. The HEWL seed crystal (1.71.11.6 mm) was glued to the seed holder in TSSG-FAST, while the HEWL seed crystal (1.9  1.2  1.7 mm) was grown at the interface of two liquids in FAST. Photographs of the setups for TSSG-FAST and FAST are presented in Figure 4. The experiment demonstrated that TSSG-FAST prevented the seed crystal from forming polycrystals. Although a number of spontaneously nucleated crystals were accumulated at the interface in TSSG-FAST (Figure 4A,B), the grown seed crystal was lifted away from the interface of the two liquids, preventing polycrystallization. Similar observations were made with TSSG-FAST using solution stirring by rotary shaker (60 rpm) (data not shown). We measured the X-ray diffraction data of the seed crystal grown by TSSGFAST, and the diffraction pattern of the obtained HEWL crystal was confirmed to be that of a single crystal. In FAST, the seed crystal formed polycrystals because the seed crystal was surrounded by spontaneously nucleated crystals at the interface of the protein-precipitant solution and the Fluorinert, resulting in adhesion of the nucleated crystals to the seed crystal (Figure 4C,D). Thus, TSSG-FAST was confirmed to be an effective tool for preventing the seed crystal from forming polycrystals, which is essential to large protein crystal growth for neutron crystallography. 3.4. Control of the Growth Direction upon Alternative Crystal Orientations. Here, two HEWL crystals with different crystal faces, that is, different orientations, are glued to the seed holders. TSSG-FAST was applied, and the shape of the HEWL crystal was observed. One crystal with an approximate size of 3.0  1.7  1.4 mm was mounted in the

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Figure 4. Photographs of the setups for TSSG-FAST and FAST. (A) Photographs of the setup for TSSG-FAST. (B) Zoomed-in view of (A). (C) A seed crystal in the FAST vessel. (D) Zoomed-in view of (C). The seed crystal appears to be polycrystallized.

seed holder with the (110) face (hereafter referred to as “(110) seed orientation”) (Figure 6A). The other crystal, with an approximate size of 2.6  2.1  1.4 mm, was mounted in the holder with the (101) face (hereafter referred to as “(101) seed orientation”) (Figure 6C). In these experiments, the HEWL crystal was grown by TSSG-FAST with stirring by rotary shaker (60 rpm). Except for the seed orientations, the crystallization condition, the amount of Fluorinert, and the growth vessels are exactly the same in this comparative analysis. Growth rates of three faces of crystals were first compared. Figure 5A depicts the schematic illustration of tetragonal HEWL crystal with established abbreviations of crystal faces. In Figure 5A, crystal sizes along the c axis, [101], [110], and [110] are denoted as the Lc-axis, L[101], L[110], and L[110], respectively. The length of L[101] can be calculated by the following equation: L[101] = Lc-axis cos β (β = 25360 ),27 where β was calculated by Palmer, Ballantyne, and Galvin from the unit cell dimensions.28 Figure 5B summarizes growth rates of (101), (110), and (110) faces, which are referred to as R(101), R(110), and R(110), respectively.

Although (110) and (110) faces must have the same growth behavior due to their crystallographic equivalence, R(110) is significantly dissimilar from R(110) in both (110) and (101) orientations. According to the previous paper,29 the value of R(101)/R(110) is approximately 2 in HEWL crystals grown by the conventional methods. However, our experiments revealed that both orientations gave a smaller value of R(101)/R(110) (1.4 for (110), and 0.9 for (101) orientations, respectively) or R(101)/R(110) (1.8 for (110), and 1.8 for (101) orientations, respectively), suggesting that TSSGFAST method produced more bulky HEWL crystals compared to that of conventional crystallization methods. To investigate the influence of the seed orientation on the crystal shape, the growth widths along [001] (along the c axis) and [110] directions [perpendicular to the (110) face (along the thickness direction)] were measured before and after crystal growth for both crystals. For the crystal with (110) seed orientation, the growth widths along the [001] and [110] directions were 0.6 and 0.2 mm; for the crystal with (101) seed orientation, the growth widths along the [001] and [110]

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Figure 5. (A) Schematic illustration of tetragonal HEWL crystal with established abbreviations of crystal faces. Crystal sizes along the c axis, [101], [110], and [110] are shown as the Lc-axis, L[101], L[110], and L[110], respectively. (B) Growth rate of individual face in the (110) orientation and (101) orientation. Growth rates (R(101, 110, 110)) of the individual faces were obtained by the following equation: (Lend(101, 110, 110) - Lstart (101, 110, 110))/2/days, where Lend and Lstart correspond to the lengths observed at the ending and starting day. Because the contact face of the seed crystal covered with glue (shown as blue columns) was assumed to inhibit the growth, the rates in the blue column were calculated by the following equation: (Lend(101, 110, 110) - Lstart (101, 110, 110))/days.

directions were 0.1 and 0.2 mm (Figure 6B,D). The degree of elongation for each crystal was calculated using the following equation: (GW110/L110)/(GW001/L001), where GW001 denotes the growth width along the [001] direction, L001 denotes the length along the [001] direction of the original crystal, GW110 denotes the growth width along the [110] direction, and L110 denotes the length along the [110] direction of the original crystal. This equation can be used to analyze the tendency of the growth direction. If a value for the equation is calculated to be less than 1, it can be judged that the crystal preferentially grew along the [001] direction rather than the [110] direction, and vice versa for values greater than 1. The crystal with (110) seed orientation gave a value for the equation of 0.72, whereas the crystal with (101) seed orientation gave a value for the equation of 3.71. These results indicated that the crystal with (110) seed orientation preferentially grew along the [001] direction, whereas the crystal with (101) seed orientation preferentially grew along the [110] direction. To confirm the reproducibility, growth rate of the other two seed crystals were also measured (Figure S2, Supporting Information). The crystal with (110) seed orientation gave a value for the equation of 1.5, whereas the crystal with (101) seed orientation gave a value for the equation of 25.5.These results also show that the crystal with (101) seed orientation tend to grow along the [110] direction. Thus, the shape of a HEWL crystal is significantly influenced by its seed orientation. The HEWL crystal with (101) seed orientation may be better for obtaining the HEWL crystal shape desired for neutron crystallography. One plausible explanation for the difference is that the contact

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Figure 6. (A) Photographs of a HEWL crystal mounted in the seed holder with the (110) face ((110) seed orientation). (B) Schematic illustration of the growth of a HEWL crystal with (110) seed orientation. In this schematic illustration, the numbers in blue indicate the crystal length along the [001] direction and the numbers in red indicate the crystal length along the [110] direction. The [001] and [110] directions are indicated in Figure 3A. The size of the seed crystal was approximately 3.0  1.7  1.4 mm. The growth widths along the [001] and [110] directions were 0.6 and 0.2 mm. The crystal with (110) seed orientation yielded a value for the equation of 0.72. (C) Photographs of a HEWL crystal mounted in the seed holder with the (101) face ((101) seed orientation). (D) Schematic illustration of the growth of a HEWL crystal with (101) seed orientation. In this schematic illustration, color coding of the numbers is the same as in (B). The size of the seed crystal was approximately 2.6  2.1  1.4 mm. The growth widths along the [001] and [110] directions were 0.1 and 0.2 mm. The crystal with (101) seed orientation yielded a value for the equation of 3.71.

face of the seed crystal is covered with glue. During crystal growth, the protein molecules are supplied to every crystal face except for the covered crystal face by the seed holder, resulting in shape anisotropy of the crystal. Consistent with this hypothesis, crystals with (110) seed orientation preferentially grew along the [001] direction, whereas crystals with (101) seed orientation preferentially grew along the [110] direction (thickness direction). An alternative explanation is the effect of the convection anisotropy around the seed crystals. Because the solution-stirring in TSSG-FAST forces the solution to flow in a specific direction, more protein molecules must be supplied to the (101) face than the (110) face in a crystal with (110) seed orientation. Conversely, more protein molecules must be supplied to the (110) face in case of a crystal with (101) seed orientation. This hypothesis is also consistent with observations of the shape of the HEWL crystals grown with an alternative seed orientation. Although further experiments must be conducted to determine the dominant effect, the overlapping mechanisms described above cannot be denied. 4. Conclusion TSSG-FAST provides a new possibility for overcoming the problems inherent in large protein crystal growth. A comparative analysis of TSSG-FAST and TSSG demonstrated

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that solution-stirring shortens the time required for crystal growth. This technique is also observed to suppress polycrystallization of the seed crystal. Notably, it was found that the shape of HEWL crystals could be successfully controlled by changing the seed orientation. Thus, we have presented the first evidence that TSSG is an effective technique for obtaining large crystals of both inorganic compounds and proteins. In addition, this method could also be applied to manipulate the protein crystals because the protein crystals are fixed on the holder, thereby allowing crystal mounting onto X-ray or neutron diffraction equipment without causing mechanical damage. The new method may overcome the difficulties of handling soft and fragile protein crystals. This novel protein crystallization technique opens the possibility of accelerating structural studies for neutron protein crystallography and subsequent structure-based drug discoveries. Acknowledgment. This work was partly supported by a Core Research for Evolution Science and Technology (CREST) grant to Y.M.; and the Science and Technology Incubation Program from the Japan Science and Technology Agency to H.M. Supporting Information Available: Figure S1: the growth widths of the other seed crystals. Figure S2: (A) Schematic illustration of the growth of a HEWL crystal with (110) and (101) seed orientation. This material is available free of charge via the Internet at http:// pubs.acs.org.

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