Improvement in Quality of Protein Crystals Grown in a High Magnetic

Article pubs.acs.org/crystal

Improvement in Quality of Protein Crystals Grown in a High Magnetic Field Gradient Akira Nakamura,†,⊥ Jun Ohtsuka,†,⊥ Ken-ichi Miyazono,†,⊥ Akihiro Yamamura,† Keiko Kubota,† Ryoichi Hirose,‡ Noriyuki Hirota,§ Mitsuo Ataka,∥ Yoriko Sawano,† and Masaru Tanokura*,† †

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan ‡ Japan Superconductor Technology, Inc., 1-5-5 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan § Advanced Key Technologies Division/Materials Processing Unit, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan ∥ National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan S Supporting Information *

ABSTRACT: Space-based microgravity environments have been utilized to obtain a highly ordered crystal because of the lack of gravity-induced convection. A superconducting magnet-based quasi-microgravity is also expected to contribute to the enhancement of the quality of protein crystals. We here report a case study on protein crystallization using fifteen kinds of samples in a magnetic field gradient, which was sufficient for magnetic levitation of water droplets. In three cases, rod-type crystals were aligned perpendicular to the crystallization plate, exhibiting magnetic orientation parallel to the direction of the magnetic field. Five proteins showed improvement in crystal quality evaluated by the resolution limit in X-ray diffraction experiments and the overall B-factor of the crystal. Our data support the idea that the reduced-gravity environment produced by a high magnetic field gradient can be used to obtain enhanced-quality protein crystals, aiding in the determination of their precise crystal structures.

1. INTRODUCTION High-precision protein structure determination by means of Xray crystallography requires high-quality crystals. However, crystal structures refined at subangstrom ( 1200 T2/m, in the cylindrical space of 40 mm ϕ (Figure 1c). The effective gravity acting on water was in the range of +0.1G to −0.1G. The crystallization temperature was 20 °C. Control experiments were also performed in parallel outside the magnetic field under the same conditions as those inside

the magnetic field, including the protein concentrations, composition of precipitant solutions, and crystallization temperature. Crystal singleness was decided based on the appearance of the crystal under a cross-polarized light microscope. 2.4. Data Collection and Processing. X-ray diffraction experiments were performed at synchrotron beamlines (SPring-8 or Photon Factory, Japan) under a 100 K nitrogen stream. To prevent ice formation during cooling, most macromolecular crystals were soaked in a liquid mixture of eight volumes of precipitant solution and 2 volumes of ethylene glycol for 10 s before the 100 K flash cooling. As one exception, a liquid mixture of seven volumes of precipitant solution and three volumes of 80% (v/v) glycerol was used instead of the ethylene glycol-containing liquid in the data collection for crystals of monomeric sarcosine oxidase. Crystals were handled with nylon CryoLoops (Hampton Research). Approximately 10 crystals were picked up in descending order of size from the crystallization drops and were used in the X-ray diffraction experiments. Several diffraction 1143

dx.doi.org/10.1021/cg2007414 | Cryst. Growth Des. 2012, 12, 1141−1150

Crystal Growth & Design

Article

Table 2. X-ray Diffraction Data Collection Statistics of MSOX Crystals data set name X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å) a

magnet-1 SPring-8 BL38B1 1.0000 50−1.95 (2.02−1.95) 5.7 (3.9) 99.4 (97.9) 20.3 (3.1) 6.6 (28.4) 0.377 19.7 P 41212 169.9 169.9 72.5

magnet-2

magnet-3

control-1

control-2

50−1.95 (2.02−1.95) 7.7 (7.5) 93.2 (91.4) 23.3 (4.2) 7.0 (36.0) 0.309 19.8

50−2.10 (2.18−2.10) 7.1 (7.0) 96.4 (96.0) 20.4 (4.6) 9.0 (35.6) 0.274 21.3

50−2.70 (2.80−2.70) 8.1 (7.8) 99.9 (100) 16.8 (4.1) 9.9 (37.5) 0.337 42.3

50−3.35 (3.47−3.35) 16.4 (15.9) 100 (100) 24.8 (13.5) 9.9 (17.2) 0.378 27.1

170.5 170.5 72.6

170.1 170.1 72.5

170.5 170.5 72.4

170.8 170.8 72.5

Values of the highest-resolution shell are in parentheses. bRsym = Σhkl|I − ⟨I⟩|/ΣhklI.

Table 3. X-ray Diffraction Data Collection and Phasing Statistics of PhAcP Crystals data set name X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å)

magnet-1

magnet-2

control-1

control-2

PF BL17A 1.0000 20−1.50 (1.54−1.50) 10.7 (10.6) 98.6 (98.3) 33.2 (7.9) 4.3 (32.9) 0.172 17.7 P3221

PF AR NW12A 1.0000 20−1.60 (1.64−1.60) 10.5 (11.0) 93.4 (96.7) 25.8 (9.8) 6.3 (33.2) 0.224 20.0

PF BL17A 1.0000 20−1.70 (1.74−1.70) 10.7 (10.3) 99.0 (94.7) 37.0 (8.1) 4.1 (30.6) 0.242 22.3

PF BL17A 1.0000 20−1.70 (1.74−1.70) 10.8 (10.9) 99.4 (100) 37.9 (8.6) 4.0 (30.0) 0.236 22.3

86.4 86.4 75.3

86.7 86.7 75.4

86.8 86.8 75.2

data set name

magnet-3

X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å) phasing statistics no. of heavy atom sites FOM (oasis/DM) automatic model building No. of residues built by ARP/wARP a

86.8 86.8 75.2 control-3

PF BL17A 2.0000 20−2.20 (2.26−2.20) 10.5 (9.9) 97.5 (95.2) 45.0 (18.7) 3.8 (10.6) 0.135 27.5 P 3221

PF BL17A 2.0000 20−2.20 (2.26−2.20) 10.7 (10.1) 95.5 (91.0) 33.5 (10.0) 5.1 (21.9) 0.255 30.5

86.5 86.5 75.4

85.9 85.9 75.4

6 0.50/0.82 175 of 182

not found


images were recorded while changing the crystal orientation, and crystal quality was evaluated comprehensively by the X-ray diffraction properties, such as the maximum resolution (⟨I⟩/σ⟨I⟩ were greater than 2.0) and the shape of diffraction spots for screening before data

collection. The diffraction intensity data sets were collected using the best crystals selected as described above. Space groups, lattice parameters, and data collection statistics were determined using the HKL2000 program suite31 or XDS.32,33 Overall B-factor values were 1144



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calculated from Wilson plots of CTRUNCATE.34 In the case of acylphosphatase, a single-wavelength anomalous dispersion (SAD) method for phasing, density modification, and subsequent automated model building of the protein three-dimensional structure were performed using SHELXC/D,35 OASIS-2006,36 DM,37 and ARP/ wARP.38,39

Table 5. X-ray Diffraction Data Collection Statistics of ST0811 Crystals data set name X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å)

3. RESULTS 3.1. Summary. This study was performed on the basis of a gravity control technique in which the weight of a protein aqueous solution was canceled out by the magnetic force acting on the solution. The strength of the magnetic field was 8−11 T. The effective gravitational forces reacting on water inside the magnetic field were from +0.1G to −0.1G, while the magnetic field and effective gravitational force for the control were 0 T and 1G, respectively. Since the much larger gradient is required for the convection-free and quasi-microgravity conditions of the diamagnetic materials (e.g., −4450 T2/m for the lysozyme crystal growth),40 our experimental environment is not convection-free or quasi-microgravity conditions for the protein crystal growth. The obtained crystals were used for the X-ray diffraction experiments, and the data collection and phasing statistics are summarized in Tables 2−6. These data were processed on the basis of our criteria, that is, ⟨I⟩/⟨σ(I)⟩ > 2 and

X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å)

magnet-1

control-1

PF AR NW12A 1.0000 20−2.16 (2.22−2.16) 5.4 (5.5) 99.2 (100) 20.9 (4.9) 5.4 (38.7) 0.202 32.6 P6322

1.0000 20−2.61 (2.68−2.61) 4.6 (3.8) 97.1 (97.1) 12.0 (2.8) 8.3 (37.7) 0.741 53.2

74.2 74.2 215.2

74.5 74.5 215.8

control-1 1.0000 50−1.59 (1.65−1.59) 3.6 (3.6) 96.3 (95.4) 30.6 (6.1) 7.6 (26.9) 0.752 19.4

55.2 55.2 221.6

54.9 54.9 222.2


a

Table 6. X-ray Diffraction Data Collection Statistics of DA06 Crystals

Table 4. X-ray Diffraction Data Collection Statistics of NDKq Crystals data set name

magnet-1 PF AR NW12A 1.0000 50−1.10 (1.14−1.10) 5.4 (2.5) 95.0 (78.8) 55.4 (6.0) 6.6 (20.5) 0.238 8.4 R3

data set name

magnet-1

magnet-2

control-1

X-ray source wavelength (Å) resolution range (Å) (outer shell) redundancya completeness (%)a ⟨I⟩/⟨σ(I)⟩a Rsym (%)a,b mosaicity Wilson B (Å2) space group cell constants a (Å) b (Å) c (Å)

PF AR NW12A 1.0000 50−1.85 (1.88−1.85) 10.7 (9.6) 99.7 (99.7) 61.6 (5.0) 5.0 (35.9) 0.300 25.9 P 321

1.0000 50−2.00 (2.03−2.00) 10.9 (10.4) 100 (100) 55.9 (7.4) 4.4 (36.8) 0.367 25.7

1.0000 50−1.90 (1.93−1.90) 11.0 (10.6) 99.9 (100) 67.0 (7.1) 4.6 (32.9) 0.389 28.4

103.8 103.8 84.9

103.7 103.7 84.8

103.8 103.8 84.8


a

33%). In the case of Nucleoside diphosphate kinase from Haloarcula sinaiiensis, Protein E, and Protein V, the crystals were not large enough for diffraction analysis. In the other seven samples, although no significant quality enhancement by the magnetic field was observed, the quality of crystals under the magnet conditions was as good as that of the control crystals. Below, we describe in detail the five case studies in which the crystal qualities of target proteins were improved by the high magnetic field-gradient environment (sections 3.2−3.6). 3.2. Monomeric Sarcosine Oxidase. Sarcosine oxidase (EC 1.5.3.1) is a flavoprotein that catalyzes the oxidative hydrolysis of sarcosine (N-methylglycine) to generate formaldehyde, glycine, and hydrogen peroxide. This enzyme is mainly classified into heterotetrameric and monomeric families. Monomeric sarcosine oxidase (MSOX) from Bacillus sp. NS129 was used for this study. Crystals of MSOX were obtained with a solution containing ammonium sulfate as a precipitant (Figures 2a, 2b). When we performed crystallization using the


a

Rsym < 40% for the highest resolution shells, and overall Rsym < 10%, defining the worthwhile resolution range for the structure analysis. The prefix “magnet-” of the data set names indicates that the data set was collected from a crystal obtained under the high magnetic field-gradient conditions. For the control crystals, the data set name was prefixed with “control-”. In three out of fifteen samples, crystals grown in the magnetic field exhibited specific orientation along the direction of the magnetic field (Figure 2). These aligned crystals gave better X-ray diffractions than those from both unoriented crystals grown in the magnetic field and control crystals obtained under gravitational conditions. Including these three, five samples showed improvement of the protein crystal quality, which was evaluated from the observed maximum resolution and the overall temperature factor (Wilson B-factor) of the crystal (the percentage of cases with quality improvement was 1145



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Figure 2. Protein crystals obtained in this study. The crystals of MSOX (a, b), PhAcP (c, d), NDKq (e, f), ST0811 (g, h), and DA06 (i, j) were grown outside (a, c, e, g, i) and inside (b, d, f, h, j) the magnetic field. Representative crystals oriented by the magnetic field are indicated by arrowheads. Scale bars (white line) indicate 50-μm lengths.

outside the magnetic field (30 ± 10 × 30 ± 10 × 220 ± 100 μm3). In addition, approximately 60% of MSOX crystals obtained in the magnetic field were standing nearly perpendicular to the crystallization plate, showing magnetic orientation. In contrast, all the control crystals were lying at the bottom of the droplet. X-ray diffraction data were collected using several crystals obtained from both the magnet and control conditions (Table 2). The magnetically oriented crystals provided better crystallographic statistical values than the unoriented crystals (data not shown) and control crystals.

solutions containing 1.9−2.3 M ammonium sulfate, single crystals were obtained within a wider concentration range of ammonium sulfate (2.1−2.3 M) under magnet conditions, while single crystals were grown only in a solution containing 2.3 M ammonium sulfate under the control conditions. The majority of the control crystals were needle-like clusters. The typical size of crystals (average of 10 crystals arbitrarily picked up from the drops of 2.3 M ammonium sulfate conditions) grown inside the magnetic field (60 ± 20 × 60 ± 20 × 280 ± 50 μm3) was significantly wider than that of crystals grown 1146



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diffracted X-rays well, whereas NDKs crystals did not provide X-ray diffractions with high enough resolution to evaluate the crystal quality. The typical size of NDKq crystals generated in the magnetic field (15 ± 5 × 15 ± 5 × 50 ± 10 μm3) was shorter than that obtained under the control conditions (15 ± 5 × 15 ± 5 × 100 ± 40 μm3). The crystals were floating or precipitated in the crystallization drops. Approximately 50% of crystals grown within the magnetic field were upright, showing an ordered magnetic orientation along the magnetic field, and the qualities of these crystals were enhanced when compared with the unoriented crystals (data not shown) and with the controls (Table 4). The 2.16 Å resolution of the magnet-1 data set is better than that of the reported data (2.59 Å resolution, PDB ID 2ZUA).23 The overall B-factor value of the magnet-1 (32.6 Å2) is better than that of the PDB data (57.6 Å2) and the control (53.2 Å2), suggesting the quality improvement of the magnetic field-grown crystal. 3.5. YjgF/YER057c/UK114-Family Protein ST0811. ST0811 protein is a member of the YjgF/YER057c/UK114 family, whose function has been unclear. Crystals of ST0811 from Sulfolobus tokodaii strain 7 were obtained with a solution containing PEG-10000 as a precipitant and these crystals were located at the bottom of the crystallization droplet (Figure 2g and h). The typical size and shape of ST0811 crystals did not differ between crystals grown and those not grown in a magnetic field (300 ± 70 × 300 ± 70 × 30 ± 10 μm3). Although the crystals grown in the magnetic field were assembled as crystal clusters (Figure 2h), the crystal quality of the diffraction data sets was higher than that of the control crystal (Table 5). We collected an X-ray diffraction data set at an atomic resolution of 1.10 Å using a crystal obtained in the magnetic field, which was greatly enhanced with respect to the resolution limit compared to the data set in the previous paper (2.00 Å resolution, PDB ID 1X25).43 Comparison of the overall B-factor values indicated that the magnet-1 (8.4 Å2) is qualityenhanced relative to the PDB data (16.0 Å2) and the control-1 (19.4 Å2). 3.6. Flap Endonuclease 1 (DA06). Flap endonuclease 1 (FEN1) is an enzyme that recognizes and removes 5′overhanging flap structures in DNA repair and RNA primers during Okazaki fragment maturation. FEN1 from Desulf urococcus amylolyticus (DA06) was crystallized in this study. The DA06 crystals were obtained with a solution containing 2 M ammonium sulfate as a precipitant (Figure 2i and 2j). The hexagonal prism crystals at a size of 70 ± 30 × 70 ± 30 × 30 ± 20 μm3 were floating and precipitating in the crystallization drops. No obvious crystal alignment was found. The resolution limits of the crystals in this study (1.85−2.00 Å) were a little higher but comparable to the previous report (2.00 Å resolution, PDB ID: 3ORY).44 The overall B-factors for the magnet data (25.7 and 25.9 Å2) were lower than those of the control (28.4 Å2) and the PDB data (34.5 Å2), although the resolution limits were not largely improved. The results indicate that the quality of DA06 crystals grown in the magnet conditions was enhanced.

The highest resolution limit in this study was observed in the magnet-1 and magnet-2 data sets, where the data were processed at 1.95 Å resolutions. Although these values were not better than that in the previous report (1.86 Å resolution, PDB ID 1ZOV),41 an improvement in the resolution limit of the magnet data was found in comparison to the control data in this study. The overall B-factors of the magnet data (19.7−21.3 Å2) were better than that of the best data in regard to the resolution limit (1ZOV, 22.2 Å2), indicating that the quality of the crystals grown in the magnetic field was enhanced. 3.3. Acylphosphatase. Acylphosphatase (EC 3.6.1.7) is an enzyme that catalyzes the hydrolysis of an acylphosphate to generate carboxylate and phosphate. Acylphosphatase from Pyrococcus horikoshii OT3 (PhAcP) was used for this study. PhAcP crystals were grown from a solution containing sodium/ potassium-tartrate as a precipitant (Figures 2c, 2d). Most crystals obtained in the magnetic field were aligned nearly perpendicular to the plate, exhibiting a specific orientation parallel to the magnetic field. These crystals (40 ± 10 × 40 ± 10 × 200 ± 100 μm3) were more than twice as thick as the control crystals (20 ± 5 × 20 ± 5 × 130 ± 60 μm3). Rodshaped PhAcP crystals were lying at the bottom of a crystallization drop and preferentially grown along the crystallographic c-axis (the long side of the crystal) in the control environment, while standing and magnetically oriented crystals were grown along the other two dimensions (ab-plane) in the magnet environment, since the depth of the crystallization droplet was limited more strictly than the breadth (Figure 2d). The oriented crystals diffracted X-rays to higher resolution than the unoriented crystals grown in the magnetic field (data not shown) and the control crystals (Table 3). The resolution limit of the magnet-1 data set (1.50 Å) was higher than that of the previously reported data (1.72 Å resolution: PDB ID 1V3Z).42 As for the data taken using an Xray wavelength of 1 Å, the overall B-factor values of the magnet data (17.7−20.0 Å2) were equivalent or better than those of the PDB data (1V3Z, 20.0 Å2) or the controls (22.3 Å2) (Table 3). We also assessed the effect of the enhancement of crystal quality through low-energy SAD phasing. SAD data were collected using X-rays at a wavelength of 2 Å and processed at up to 2.2-Å resolution (magnet-3 and control-3 in Table 3). We searched for the positions of six atoms from which anomalous signals were expected to be detected by the 2-Å X-rays, because the crystal structure of PhAcP contains two potassium ions (f ″ = 1.7e at 2 Å), two chloride ions (1.1e), and two sulfur atoms (0.9e) (PDB 1V3Z).42 Phasing statistics are also listed in Table 3, and graphs representing the qualities of crystals are shown in the Supporting Information Figure S1. Although both data sets were processed up to the same resolution of 2.2 Å, the quality of the control-3 data set was not sufficient to determine the phase angles of their crystal structure factors. In contrast, the phase angle calculation and subsequent automated model building were successful when we used the magnet-3 data set. 3.4. Nucleoside Diphosphate Kinase. Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the transfer of the γ-phosphate group from nucleoside triphosphates to nucleoside diphosphates. GTP produced by GDP-forming succinyl-CoA synthetase in the citric acid cycle is converted to ATP by the reaction of NDK. NDK from Haloarcula quadrata (NDKq) and Haloarcula sinaiiensis (NDKs) were examined in this study. NDKq crystals were obtained with a solution containing polyethylene glycol (PEG)4000 as a precipitant (Figure 2e and f), and these crystals

4. DISCUSSION Our study strongly supports that the use of a reduced-gravity environment based on magnetic field gradients improves protein crystal quality.13 The crystals obtained in the magnetic field were frequently well-separated and grown at a larger size compared with the control crystals, indicating that our experimental environment could reduce the production of 1147



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our crystallization experiments, the crystal quality was improved not only by the usage of a highly viscous PEG solution (NDKq and ST0811) but also using a solution with a high concentration of salt (MSOX, PhAcP, DA06). The results imply that salt-based conditions also contribute to quality improvement of protein crystals in the high magnetic field. There are three cases in which the crystals obtained under magnet conditions exhibited specific orientations (MSOX, PhAcP, and NDKq). More than half of these crystals were standing nearly perpendicular to the crystallization plate and parallel to the direction of the magnetic field (Figure 2b, d, and f). The magnetic orientation of protein crystals occurring even without paramagnetic substances such as ferric and nickel atoms is thought to be mainly induced by the susceptibility anisotropy of diamagnetic protein molecules with the magnetic anisotropy of crystallographic unit cells.9,48−50 The magnetic stabilization energy is defined as ΔE = (1/2μ0)|Δχ|VB2, where μ0 is the absolute permeability of vacuum, Δχ and V are the anisotropy of diamagnetic susceptibility and the volume of a crystal, respectively, and B is the magnetic flux density.50 Since the absolute values of diamagnetic substances such as water and proteins are small, crystals are required to be large enough and to be floating in the crystallization drop in order to overcome the thermal fluctuations (ΔE ≫ kT, where k is the Boltzmann constant and T is the absolute temperature) and to avoid physical stress by the contact with a vessel, respectively.9 The unaligned crystals of MSOX, PhAcP, and NDKq are thought to be precipitated before responding to the magnetic field. The correlation between the magnetic orientation and the crystalline quality improvement can be explained by the fact that the mosaic blocks would be better oriented along the magnetic field, resulting in a decrease in crystal mosaicity.12,19 The increase of viscosity by oriented crystals in the high magnetic field45 may also contribute to improvement of crystal quality. Though the detailed mechanism of crystal quality enhancement remains to be clarified, magnetically oriented protein crystals have shown better diffraction quality so far.13,19 Therefore, further investigations measuring or estimating the macromolecular magnetization and examining the relationship between the diamagnetic anisotropy and magnetic orientation would be enormously valuable in predicting whether or not the crystal quality of target macromolecules has the potential to be increased using a magnetic field. From our results, we would expect to find a correlation between space groups and the magnetic orientation of crystals. The three protein crystals that exhibited magnetic orientation in this study belong to the space groups of P41212, P3221, and P6322. Previous works have reported that crystals of hen egg-white lysozyme (P43212/P21/ P212121),7,9,19,51 bovine pancreatic trypsin inhibitor (P6422/ P6322),8 porcine pancreatic α-amylase (P212121),8 bovine pancreatic ribonuclease A (P3221),9 sperm whale metmyoglobin (P21),9 snake muscle fructose-1,6-bisphosphatase (P3121),13 and thaumatin (P41212)52 showed magnetic orientation. The space groups of these crystals characteristically and absolutely include a screw axis (axes). Of all possible space groups for protein crystals, the percentage possessing a screw axis is calculated to be 63.1% (41/65). The proportion of entries in the PDB that belong to space groups containing a screw axis is calculated to be 77.7% of X-ray crystal structures (based on the data of June seventh, 2011). Therefore, although approximately three-quarters of protein crystals have a crystallographic screw axis, it could be meaningful that all reported and reporting protein crystals exhibiting the magnetic

crystal nuclei and affect crystal growth. X-ray diffraction experiments revealed that the crystal quality was improved in one-third of the samples. For the five samples described above in details (sections 3.2−3.6), the crystal qualities, which were judged by the X-ray highest resolution limit and overall Bfactor, of the magnetic field-grown crystals were enhanced compared with those of the control crystals. The resolution limit is a readily understood indicator of the precision of the structure analysis. The overall B-factor values indicate the degree of crystal internal order and are essentially independent of crystal size; these values have been used to evaluate the crystal quality.3,20 The crystals grown in the magnetic field showed better X-ray resolutions than those reported in the literatures for four cases (PhAcP, NDKq, ST0811, and DA06). All five samples including MSOX displayed improved overall Bfactor values. In the other ten samples, the good effects induced by the reduced-gravity environments described above must have taken place in crystals. However, inherent structural heterogeneity of the proteins attributed to disordered loop structures would be a predominant factor in defining the crystal quality. Therefore, it is presumed that no improvement was detected in the X-ray diffraction qualities. It has also been reported that the space-based microgravity environment is not always effective for improving crystal quality.17 Comparing the diffraction data (Tables 2−6), it should be noted that crystals grown in the magnetic field give better values not only for the maximum resolution and overall B-factor but also for the Rsym factor and crystal mosaicity as reported previously.19 As for PhAcP, the low-energy (6.2 keV) SAD phasing for the 2.2-Å resolution data set was successful only when using the data from a crystal obtained in the magnetic field. The major differences between the two SAD data sets of PhAcP (magnet-3 and control-3) were in the calculated values of Rsym and crystal mosaicity, and the values of these parameters indicated that the magnet-3 data set was better than the control-3 data set. In addition, the anomalous difference signals of the crystal grown under the magnetic field were significantly stronger than those of the control crystal (Supporting Information Figure S1). It seems clear that these crystallographic parameters are critical for the determination of phases by means of the SAD method, in which the precise measurement of diffraction intensities in an X-ray diffraction experiment is necessary to detect subtle differences of structure factors derived from anomalous signals. Although only one case of application to SAD phasing was reported here and only onethird of the samples showed improvement in X-ray diffraction properties, these results may imply that crystallization in the high magnetic field gradient is a useful method to easily obtain better crystals, leading to high-throughput protein structure determination. It has been reported that the viscosity of a protein aqueous solution with undissolved microcrystals is increased by application of a high magnetic field.45 Increased viscosity is expected to contribute to damping the natural convection, resulting in a decrease of the protein crystal growth rate.4 This could be explained by the previous experimental results that the depletion zone around a protein crystal remains undisturbed in a highly viscous solution of PEG-8000.18 In addition, highquality protein crystals were obtained in high-viscosity solutions under space-based microgravity conditions,46,47 in which the combination of a highly viscous and microgravitational environment was accomplished in the crystallization solution. Concerning the compounds that were used as precipitants for 1148



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Notes

orientation belong to space groups possessing a screw axis. A pillar-type nonspherical crystal may be grown for protein crystals containing a screw axis, which have some benefit for the magnetic orientation or simply make us notice the crystal alignment. Though ST0811 (R3) and DA06 (P321) crystals, whose shapes were triangular and hexagonal prisms, respectively, seemed not to show a magnetic orientation, these crystals certainly exhibited an improved quality grown under the magnetic field gradient, likely by the effect of the reducedgravity environment. In the case of Gnk2, the crystal includes a screw axis but the crystal symmetry is cubic with isotropic properties. Therefore, Gnk2 crystals did not exhibit magnetic orientation inherently. This is consistent with previous reports, in which cubic crystals (F432) of horse spleen ferritin did not show magnetic orientation.6,52 Consequently, we suggest that the presence of a screw axis in anisotropic space groups for protein crystals may strengthen the magnetic anisotropy and may be somewhat involved in crystal quality improvement through magnetic orientation, whereas the magnetic anisotropy of a protein molecule and a crystal would contribute mainly to the magnetic orientation. However, we also obtained conflicting results that crystals of some proteins (ST1710, Protein L, Protein N, Protein P, and Protein S) did not exhibit magnetic orientation, although they satisfied the screw-axis condition. The crystals of these samples, together with ST0811 and DA06, might have insufficient anisotropy of magnetic susceptibility to respond to the magnetic field. Further statistical analyses based on a large number of experiments with various proteins together with in vitro and in silico studies of diamagnetic anisotropy could contribute to the validation of this expectation.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the beamline staff at both the Photon Factory and SPring-8 for their kind help with data collection. Synchrotron radiation experiments were performed at beamlines BL-17A and AR-NW12A of the Photon Factory (Tsukuba, Japan) (Proposal No. 2006S2-006), and at beamline BL-38B1 of SPring-8 (Harima, Japan) (Proposal No. 2006A1721). This work was supported by the System Development Program for Advanced Measurement and Analysis (Program-S) of the Japan Science and Technology Agency (JST) and in part by the National Project on Protein Structural and Functional Analyses and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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5. CONCLUSION We here reported case studies on protein crystallization in a magnetic field gradient. Five cases showed improvement in crystal quality evaluated by the X-ray maximum resolution limit and the overall B-factor of the crystal. In three of these five cases, the crystals exhibited magnetic orientation, resulting in remarkable improvement of the crystal quality. The reducedgravity conditions generated by magnetic force have an advantage over gravity-free conditions in space in the production of high-quality crystals because the magnetic field can orient crystals. Protein crystallization in a high magnetic field gradient therefore is considerably beneficial to yield moreordered quality-enhanced crystals of various proteins of interest.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information on the proteins used in this study is listed in SI Table S1 and Rsym, ⟨I⟩/⟨σ(I)⟩, and mean anomalous difference values versus resolution shells for PhAcP data sets are shown in SI Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Address: 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657. Tel: +813-5841-5165. Fax: +81-3-5841-8023. E-mail: amtanok@mail. ecc.u-tokyo.ac.jp. Web: http://fesb.ch.a.u-tokyo.ac.jp/. Author Contributions ⊥

These authors contributed equally to this work. 1149



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Improvement in Quality of Protein Crystals Grown in a High Magnetic

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