ISSN 10637745, Crystallography Reports, 2011, Vol. 56, No. 5, pp. 884–891. © Pleiades Publishing, Inc., 2011. Original Russian Text © I.P. Kuranova, E.A. Smirnova, Yu.A. Abramchik, L.A. Chupova, R.S. Esipov, V.Kh. Akparov, V.I. Timofeev, M.V. Kovalchuk, 2011, published in Kristal lografiya, 2011, Vol. 56, No. 5, pp. 944–951.
CRYSTAL GROWTH
Crystal Growth of Phosphopantetheine Adenylyltransferase, Carboxypeptidase T, and Thymidine Phosphorylase on the International Space Station by the Capillary CounterDiffusion Method I. P. Kuranovaa, E. A. Smirnovaa, Yu. A. Abramchika, b, L. A. Chupovab, R. S. Esipovb, V. Kh. Akparovc, V. I. Timofeeva, and M. V. Kovalchuka, d a Shubnikov
Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 119333 Russia email:
[email protected] b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117871 Russia c Scientific Center of Russian Federation, Research Institute for Genetics and Selection of Industrial Microorganisms, Pervyi Dorozhnyi proezd 1, Moscow, 113545 Russia d National Research Center Kurchatov Institute, Moscow, Russia Received April 27, 2011
Abstract—Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counterdiffusion method in the Japanese Experimental Module Kibo on the International Space Station. The highresolution Xray diffraction data sets suitable for the determination of highresolution threedimensional structures of these proteins were collected from the grown crystals on the SPring8 syn chrotron radiation facility. The conditions of crystal growth for the proteins and the datacollection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth. DOI: 10.1134/S1063774511050154
INTRODUCTION Protein crystal growth in microgravity is one way to improve the quality of diffraction patterns of crystals [1, 2]. Due to the absence of convective flows in microgravity, the mass transport to growing crystals occurs primarily through diffusion. The absence of sedimentation and the spherical geometry of the diffu sion field are favorable for the growth of most isomet ric crystals. In the absence of the force of gravity, a sta ble concentration gradient of protein molecules and admixtures is established in solution around the grow ing crystals, which, on the one hand, enhances the segregation of admixtures and, on the other hand, enables protein molecules to be included in the crystal lattice in the optimal orientation [3, 4]. The stable concentration gradient around crystals in microgravity reduces the probability of secondary nucleation, thus preventing crystal intergrowth and the formation of druses. In some cases single crystals were obtained in microgravity instead of twins that grew on Earth [5]. Protein crystal growth in microgravity is performed in specially designed equipment but with the use of the same methods as those used for crystallization on
earth. However, it was shown that the vapordiffusion method most widely used in unit gravity has some drawbacks in microgravity. Thus, due to the presence of the free surface, convective flows (Marangoni con vection) are induced around crystals in drops with increasing crystal sizes, which impairs the quality of the crystals [6]. Hence, the freeinterface diffusion through the interface between the protein and precipitant solu tions and the counterdiffusion are the methods of choice for crystallization experiments in microgravity. We used the freeinterface diffusion method for the protein crystal growth in the Modul’1 protein crystal lization apparatus on the International Space Station (ISS) [7, 8]. In this study we describe the crystal growth of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPAT Mt), thymi dine phosphorylase (TP) from Escherichia coli, car boxypeptidase T (CPT) from Thermoactinomyces vul garis and its mutant forms, and complexes of these proteins with functional ligands and inhibitors by the capillary counterdiffusion method in the Kibo exper imental module of the Japan Aerospace Exploration Agency (JAXA) on the ISS. The experiments were
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performed with the participation of the JAXA researchers. MATERIALS AND METHODS The Preparation of Phosphopantetheine Adenylyl transferase from Mycobacterium tuberculosis. Recom binant phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPAT Mt) was prepared according to a procedure described in [9]. The aviru lent producing strain E. coli ER2566/pER_PPAT was cultivated in the YT medium containing ampicillin (50 mg/mL) and grown at 37°С. The cells were sepa rated by centrifugation. After ultrasonic disintegra tion, the homogenate was centrifuged. The superna tant containing the target protein was successively fractionated on Sepharose Q XL and Sepharose Q HP with a NaCl gradient. The final purification of the pro tein was performed by gel filtration on a Sepharose S 200. The protein solution in a HEPES/HCl buffer, pH 8, was stored in the frozen state at –80°. Recombinant carboxypeptidase T from Thermoacti nomyces vulgaris (CPTwt) was prepared by cloning the CPT gene into E. coli followed by the renaturation of the protein from inclusion bodies and purification by affinity chromatography on paminobenzylsuccinic acid coupled to activated Sepharose [10]. The procpT mutant genes were produced by the standard PCRbased targeted mutagenesis. The pres ence of mutations was confirmed by gene sequencing. The mutant forms were expressed in E. coli BL21(DE3)pLysS cells according to the manufac turer’s instruction (Novagen). The activation of proCPT5 was carried out with the use of subtilisin. The activated CPT5 was purified on a CABS Sepharose affinity column and then concentrated using a Millipore membrane filter [11]. The CPT5 protein had the following mutations in the primary specificity site: G215S, A251G, T257A, D260G, T262D. Recombinant thymidine phosphorylase from E. coli was produced in the bacterial strain E. coli BL21(DE3)/pERThy1. In the first stage of purifica tion, the supernatant was twice precipitated with ammonium sulfate. The further purification of the protein was performed by anionexchange chroma tography on the sorbent Sepharose Q HP and by hydrophobic chromatography on the sorbent Phenyl Sepharose HP [12]. Initial crystallization conditions for all proteins were found with the use of the vapordiffusion method. These conditions were modified to use the capillary counterdiffusion method by varying the protein and precipitant concentrations. Crystallization by the capillary counterdiffusion method. The crystallization was performed in glass capillaries (Confocal Science Inc.) 60 mm in length and 0.3 or 0.5 mm in inner diameter plugged with sili cone tubes filled with agarose gel. The crystallization CRYSTALLOGRAPHY REPORTS
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Capillary Tube filled with agarose gel Fig. 1. Capillary containing a protein solution and plugged with a silicone tube filled with agarose gel.
conditions were optimized in capillaries with an inner diameter of 0.3 and 0.5 mm for experiments in micro gravity and the control experiment on earth, respec tively. To fill a capillary, a 3–5 µl (a capillary with a diam eter of 0.3 mm) or 8–10 µl (a capillary with a diameter of 0.5 mm) drop of the protein solution was placed onto a siliconized glass surface and the solution was sucked into the capillary by touching the drop with the capillary tip. The end of the capillary opposite the end filled with the solution was hermetically sealed by placing it into plasticine. A silicone tube 15 mm in length filled with 1% agarose gel was attached to the end of the capillary containing the solution (Fig. 1). In the experiments on earth, the precipitant solution was placed in a test tube (the socalled GT method) [13]. The silicone tube was cut with a sharp razor blade to the desired length, and the capillary with the gel tube was placed into a screwcap test tube containing 1 ml of the precipitant solution. The gel tube was dipped completely into the solution (Fig. 2). The capillaries were withdrawn from the test tubes every 4–6 days, and the nucleation and crystal growth were observed with a microscope. Pictures of the crystals in capillar ies were taken with a digital camera connected to the microscope. Filling of capillaries and the assembly of a JAXA Crystallization Boxes (JCB) for launching to the ISS. To perform the crystallization experiment in micrograv ity, we prepared the protein and precipitant solutions. The compositions of the solutions and the concentra tions of components corresponded to the optimal crystal growth conditions. The preflight filling of capillaries with the solu tions, the assembly, the hermetic sealing, and the packing of crystallization boxes produced by JAXA for launching to the ISS were performed by JAXA researchers according to the protocol developed at JAXA [14]. A glass capillary 0.5 mm in diameter and 60 mm in length was loaded with a protein solution to the height of 30 mm, one end of the capillary was sealed with plasticine, and a silicone tube filled with 1% agarose gel (presoaked in the precipitant solution for one day) was attached to another end of the capillary. Each of two 180µl plastic cylinders connected with each other was filled with onehalf of the precipitant solution. Then a capillary with an attached gel tube was filled with the protein solution and placed in each cylinder.
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Visual inspection of the crystal morphology and the location of crystals in capillaries. After the space flight, the boxes containing capillaries were viewed and their photos were taken with an Olympus microscope with out being taken out of their plastic packages. Then the boxes were placed back in their containers and trans ported to the SPring 8 synchrotron radiation facility (Japan), where the boxes were opened and Xray dif fraction data were collected. Preparation of stabilizing and cryoprotectant solu tions for harvesting crystals and Xray diffraction data collection. The corresponding stabilizing and cryopro tectant solutions were prepared for each type of crys tal. The stabilizing solutions for harvesting crystals contained a higher percentage of solvent than the mother liquor in which the corresponding crystals grew. Solutions for the flash freezing of crystals prior to the data collection contained components at the same concentrations as the stabilizing solutions and, in addition, 20–25% glycerol. Xray data collection at the SPring 8 synchrotron radiation facility. To harvest crystals from capillaries, the latter were cut under a microscope in such a way that the crystal chosen for Xray data collection was in the shorter part of the capillary. The crystal was taken out from the capillary by carefully washing it out into a drop of the harvest solution using a flow of this solu tion through a pipette tip tightly pressed to the capil lary. The crystals were picked up from the harvest solu tion with a nylon loop on a magnetic pin, transferred for a few seconds into a glycerolcontaining cryopro tectant solution, and then frozen in nitrogen vapor. The Xray data sets were collected at 100 K at the SPring8 synchrotron radiation facility (Japan) at the BL41XU beamline equipped with MX225HE or QUANTUM315 CCD detectors.
Fig. 2. Geltube (GT) method of crystallization; a capil lary with a gel tube placed in a test tube containing a pre cipitant solution.
Bottom of the cylinder
Cylinder containing a precipitant Capillary
Fig. 3. JCB, which accommodates six capillaries and is enclosed in a sealed plastic bag.
RESULTS AND DISCUSSION
In this case the precipitant solution was partially dis placed from the cylinder and filled the whole volume. The lower open ends of both cylinders (bottoms of the cylinders) were closed with plugs provided with micro holes for the removal of air and an excess of the solu tion and then thoroughly hermetically sealed with glue. All six cylinders with capillaries were placed in a polyethylene case containing a small amount of water. The cases were hermetically sealed and placed in con tainers (Fig. 3). The containers were delivered to the space launching center in a thermal insulated bag. The temperature was controlled with the use of plastic packages containing hexadecane and heptadecane. The crystal growth conditions for the proteins and their complexes with functional ligands and inhibitors, which were used in the space flights and resulted in the highest quality crystals, are given in Table 1.
The aim of experiments performed in collaboration with JAXA was to grow protein crystals of high diffrac tion quality in microgravity by the counterdiffusion method. The main distinguishing feature of the counterdif fusion method developed by GarciaRuiz and Moreno [15] is that a protein solution is mixed with a precipi tant solution for crystallization not directly through the interface between the protein and precipitant solu tions but through a layer of gel placed between these solutions. The crystallization occurs in a capillary. Due to the presence of a gel plug, the rate of diffusion of the precipitant into the protein solution is lower and convective flows are reduced even in unit gravity [4]. The oppositely directed concentration gradients of the protein and precipitant solutions are established along the capillary. The crystals that appear at different dis tances from the entry to the capillary grow at different proteintoprecipitant concentration ratios, which is
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Table 1. Crystal growth conditions for proteins in microgravity during the JAXA1–JAXA3 space flights Protein, concentration PPAT Mt, concentration was 12 mg/ml PPAT Mt/CoA, concen tration was 12 mg/ml PPAT Mt/DPCoA, con centration was 16 mg/ml PPAT Mt/ATP (I), con centration was 12 mg/ml PPAT Mt/ATP (II), con centration was 10 mg/ml
Composition of the protein so lution
Composition of the precipitant solution 40 mM cacodylate, pH 5.5, 20 mM MgCl2, 20 mM [Co(NH3)6]Cl3, 15% MPD 20 mM cacodylate, pH 5.5, 5 mM MgCl2, 0.075 M NaCl, 12% MPD, 5 mM HEPES, pH 8.0, 20 mM cobalt hexamine, 14 mM CoA 20 mM cacodylate, pH 5.5, 5 mM MgCl2, 0.075 M NaCl, 15% MPD, 5 mM HEPES, pH 8.0, 20 mM cobalt hexamine, 14 mM DPCoA 20 mM cacodylate, pH 5.5, 5 mM MgCl2, 0.075 M NaCl, 12% MPD, 5 mM HEPES, pH 8.0, 20 mM cobalt hexamine, 14 mM ATP 0.1 M NaAc, pH 5.0, 10 mM MgCl2, 5 mM HEPES, pH 8.0, 0.075 M NaCl, 14 mM ATP, 1.1 M ammonium sulfate 1.6 M (NH4)2SO4, 5% MPD, 50 mM Mes/NaOH, pH 6.0 1.4 M (NH4)2SO4, 5% MPD, 50 mM Mes/NaOH, pH 6.0, 18 mg/ml ZLLys 1.4 M (NH4)2SO4, 5% MPD, 50 mM Mes/NaOH, pH 6.0, 2.4 M BocLLeu 1.2 M (NH4)2SO4, 250 mM NaCl, 5% MPD, 1 mM CaCl2, 1 mM ZnAc, 50 mM Mes/NaOH, pH 6.0 1.2 M (NH4)2SO4, 5% MPD, 50 mM Mes/NaOH, pH 6.0, 1mM CaCl2, 18 mg/ml ZLLys 1.4 M (NH4)2SO4, 5% MPD, 50 mM Mes/NaOH, pH 6.0, 1 mM CaCl2, 1 mM ZnAc, 2.4 mg/ml BocLLeu 25% (NH4)2SO4, 0.1 M sodium citrate, pH 5.5, 0.04% NaN3
10 mM HEPES, pH 8.0, 0.15 M NaCl, 1 mM DTT 10 mM HEPES, pH 8.0, 0.15 M NaCl, 14 mM CoA, 1 mM DTT 10 mM HEPES, pH 8.0, 0.15 M NaCl, 14 mM DPCoA, 1 mM DTT 10 mM HEPES, pH 8.0, 0.15 M NaCl, 14 mM ATP, 1 mM DTT 10 mM HEPES, pH 8.0, 0.15 M NaCl, 14 mM ATP
CPT wt, concentration was 10 mg/ml CPT wt/ZLLys, con centration was 10 mg/ml CPT wt/BocLLeu, con centration was 10 mg/ml CPT 5, concentration was 11.4 mg/ml
250 mM NaCl, 10 mM Mes/NaOH, pH 6 250 mM NaCl, 1mM CaCl2, 10 mM Mes/NaOH, pH 6.0 250 mM NaCl, 1mM CaCl2, 10 mM Mes/NaOH, pH 6.0 250 mM NaCl, 1 mM CaCl2, 1 mM ZnAc, 10 mM Mes/NaOH, pH 6.0 CPT 5, ZLLys, concen 250 mM NaCl, 1 mM CaCl2, tration was 11.4 mg/ml 1 mM ZnAc, 10 mM Mes/NaOH, pH 6.0 CPT 5/BocLLeu, con 250 mM NaCl, 1 mM CaCl2, centration was 11.4 mg/ml 1 mM ZnAc, 10 mM Mes/NaOH pH 6.0 Thymidine phosphorylase 0.1 M KH2PO4, pH 7.3, from E. coli, concentra 0.04% NaN3 tion was 10 mg/ml Thymidine phosphorylase 0.025 M KH2PO4, pH 7.3, 0.04% NaN3, 2 mM from E. coli/inhibitor 1, concentration was 17 mg/ml 3'NH22'F2',3'ddt Thymidine phosphorylase 0.05 M KH2PO4, pH 7.3, from E. coli/inhibitor 2, 0.04% NaN3, 4 mM concentration was 35 mg/ml 3'NH22',3'ddt
30% (NH4)2SO4, 0.1 M sodium citrate, pH 5.5, 0.04% NaN3, 2 mM 3'NH22'F 2',3'ddt 25% (NH4)2SO4, 0.1 M sodium citrate, pH 5.5, 0.04% NaN3, 4 mM 3'NH22',3'ddt
why several growth conditions can be tested in one capillary. To perform experiments on crystal growth by the counterdiffusion method in microgravity, Spanish researchers constructed the Granada Crystallization Box (GCB) [16]. In the GCB crystals were grown directly in thinwalled Xray capillaries. One end of the capillary was sealed with vacuum grease, and another end was inserted into a buffered agarose gel, which was placed in a plastic box and covered with a precipitant solution. The plastic box accommodated six capillaries. CRYSTALLOGRAPHY REPORTS
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Crystal size, mm 0.15–0.25 0.15–0.30
0.20–0.45
0.20–0.35
0.05–0.20
0.35–0.45 0.15–0.35 0.20–0.35 0.1–0.4
0.3–0.4
0.3–0.4
0.15–0.4
0.15–0.5
0.5
Japanese researchers modified the crystallization device for the JCB counterdiffusion method 14]. In this device, thickwalled glass capillaries with a diam eter of 0.3 or 0.5 mm are used instead of fragile Xray capillaries. Agarose gel is polymerized in a long sili cone tube, and then the gel tube is cut into pieces of a length of up to 1.5 mm. The piece of the gel tube is attached to the end of the capillary. The capillary with the attached tube (through which the precipitant should diffuse) is dipped into a small volume of the precipitant solution in a cylindrical tube and both ends of the latter are thoroughly hermetically sealed. The length of the silicone tube attached to the capillary can be varied, which makes it possible to control the onset
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In this study the initial screening of protein crystal lization conditions was performed by the hanging drop vapordiffusion method. The crystallization con ditions thus found were optimized as applied to the counterdiffusion method in capillaries with a diame ter of 0.3 mm. A protein solution (3 µl) was loaded in each capillary. Then the capillaries were placed in a screwcap test tube containing 0.5–1.0 ml of the pre cipitant solution. The crystallization experiments on the ISS were carried out in the Japanese Experiment Module Kibo of the Japan Aerospace Exploration Agency with the use of the JCB [14]. Crystals of PPAT Mt and its complexes with func tional ligands, carboxypeptidase T (CPT wt) and its mutant form CPT5 with the incorporated primary specificity site of carboxypeptidase B, thymidine phosphorylase from E. coli, and complexes of these proteins were grown by the counterdiffusion method on the ISS during the JAXA1–JAXA3 space flights.
(a)
Table 1 lists the proteins and their complexes with ligands and the crystallization conditions in which Xray quality crystals were obtained. The photos of some crystals are shown in Figs. 4–7.
(b) Fig. 4. Crystals of PPAT Mt; (a) free enzyme; (b) the com plex with DPCoA.
of crystallization. This modified method has a number of advantages. Since a gel tube is attached to each cap illary, different precipitants can be used for different capillaries. Due to the small volume of the gel in the tube, a smaller volume of the precipitant solution can be used in experiments. The latter fact is particularly important when it is necessary to add an expensive ligand to the precipitant solution for the preparation of crystals of protein complexes with noncovalently bound ligands. To estimate changes in the concentrations of the protein and the precipitant at any point in the capillary depending on the time, H. Tanaka et al. developed the 1D simulation program [13]. Differential equations describing the onedimensional diffusion process were used as a mathematical model for the diffusion of pro teins and precipitants in gel and in a capillary. The dif fusion coefficient is evaluated from the data on the molecular weights and the initial concentrations of the protein and the precipitant. The concentrations of the protein and the precipitant at which crystals appeared, which were thus estimated, can be used as the starting crystallization conditions in subsequent experiments.
The protein PPAT Mt is involved in the coenzyme A (CoA) biosynthesis by catalyzing the penultimate step of this process resulting in the formation of the dephosphocoenzyme A (DPCoA) from 4’phospho pantetheine and ATP. The PPATcatalyzed reaction is the key step in the synthesis. Its rate is controlled by the coenzyme A, which is the final product of the cycle. The latter inhibits the enzyme by forming a complex with PPAT. Since coenzyme A is necessary for vital functions of the mycobacterium causing tuberculosis, PPAT is a convenient target for the design of antituberculosis drugs, particularly taking into account the fact that the coenzyme A biosynthesis in mammalian organisms is performed by the bifunc tional enzyme that differs from bacterial PPAT. Crys tals of both the free enzyme and its complexes with the natural inhibitor (coenzyme A), the reaction product (dephosphocoenzyme A), and the substrate (ATP) were grown on the ISS. Adenosine triphosphate is one of the substrates of the PPATcatalyzed reaction. The complex PPAT Mt/ATP was grown with the use of two different pre cipitants (ammonium sulfate and 2methyl2,4pen tanediol (MPD) in the presence of cobalt hexamine). A comparison of the structures of the crystals grown in the presence of different precipitants will give infor mation on how the weakening of hydrogen bonds due to the presence of the organic solvent (MPD) influ ences the character of intermolecular bonds in the crystal lattice. The crystals of the complex PPAT Mt/ATP were also prepared in the presence of magne sium ions, which are necessary for the reaction to occur.
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(a)
(a)
(b) (b) Fig. 5. Crystals of PPAT Mt; (a) the complex with CoA; (b) the complex with ATP.
Fig. 6. Crystals of carboxypeptidase T: (a) CPT wt; (b) CPT 5.
Data on the structure of the complex PPAT/CoA are important also for understanding the mechanism of enzyme inhibition. Coenzyme A is not directly involved in the PPATcatalyzed reaction, but it is a natural inhibitor of the enzyme. When the concentra tion of CoA increases, it forms a complex with PPAT, thus interrupting the biosynthesis in the penultimate step.
can serve as the structural basis for the targeted search for specific inhibitors of the enzyme. The latter are potential antituberculosis drugs. Recombinant bacterial carboxypeptidase T (CPT) from Thermoactinomyces vulgaris is of particular inter est as a convenient enzyme for investigating the struc tural basis for the specificity of this enzyme family. The rational change in the specificity is not only important in the theoretical aspect for an understanding of the enzyme catalysis, but it is also an important problem of engineering enzymology. The structure of the active site of CPT is similar to that of mammalian carboxypeptidases A and B, but it is characterized by a wider specificity and cleaves, though at different rates, the Cterminal positively charged, negatively charged, and hydrophobic amino acid residues. The mutant CPT5 has five replacements in the substratebinding site S1’, resulting in it being structurally similar to the site S1’ of carboxypeptidase B. However, an investigation into the properties of the mutant form showed that, in spite of the replacements, CPT5 retains the carboxypeptidase T specificity. This result confirms the hypothesis that the specificity of the enzyme depends not only on the nature of the
Earlier, in the course of screening of the protein crystallization conditions by the vapordiffusion method, we have grown crystals of the complex PPAT Mt/CoA and determined its threedimensional struc ture at 2.1 Å resolution [9]. The Xray diffraction data collected in the present study made it possible to increase the resolution of the crystal structure to 1.59 Å. The determination of the threedimensional struc ture of free PPAT and its complexes with substrates and reaction products will provide insight into the conformational changes attendant to the enzymatic reaction, which is necessary for an understanding of its mechanism. In addition, data on the threedimen sional structure of the target protein and its complexes CRYSTALLOGRAPHY REPORTS
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Fig. 7. Crystals of free thymidine phosphorylase from E. coli.
aminoacid residues directly interacting with the sub strate, but also on the nature of the residues distant from the bound substrate. To compare the arrangement of the side chains of the aminoacid residues that are involved in the active
sites S1’ of both forms of the enzyme, Xray quality crystals of the complexes of CPTwt and CPT5 with leucine and lysine derivatives (BOCLLeu and ZLLys, respectively) were grown. The determination of the threedimensional structures of CPT and its mutant forms having replacements in the substratebinding site and its environment will make it possible to reveal distant structural determinants of the specificity of the enzyme. The crystals of CPT5 grown earlier on earth diffracted to 2.1 Å resolution [11]. The crystals of CPT5 grown in microgravity diffracted to 1.09 Å reso lution. Thymidine phosphorylase catalyzes the catabolic phosphorolysis of pyrimidine nucleosides in bacterial cells. This enzyme is used in the synthesis of purine and pyrimidine deoxynucleotides, many of which can be used as drugs. Thymidine phosphorylase plays an important role in cells by stimulating new bloodvessel growth. High levels of this enzyme were detected in certain tumor cells. Hence, there is an active search for specific inhibitors of thymidine phosphorylase. The crystals were grown by modifying the conditions reported in [17]. In the present study, crystals of the free protein and complexes of TP with two inhibitors—3amino 2fluoro2,3dideoxythymidine (inhibitor 1) and 3
Table 2. Characteristics of protein crystals grown in microgravity and Xray data collection statistics Resolution, Å Protein crystals
Space group
earth grown
space grown
crystals are absent 1.8
1.6
H32
1.4
H32
PPAT/dpCoA
1.8
1.5
H32
PPAT/ATP
1.8
1.5
H32
1.81
P321
PPAT PPAT/CoA
PPAT/ATP + Mg
>5
CPT5
1.4
1.09
P6322
CPT5/ZLLys
1.9
1.4
P6322
CPT5/BOCLLeu
1.7
1.4
P6322
1.21
P6322
CPT wt/ZLLys
crystals are absent CPT wt/BOCLLeu crystals are absent TP >2
1.12
P6322
1.52
P43212
TP/inhibitor 1
1.7
P43212
1.85
P43212
TP/inhibitor 2
>2 crystals are absent
Unitcell parameters, Å, deg
Complete Rmerge ness of da (I), % ta, %
a = b = 98.67, c = 113.85, α = β = 90.00, γ = 120.00 a = b = 98.88, c = 114.86, α = β = 90.00, γ = 120.00 a = b = 99.22, c = 115.85, α = β = 90.00, γ = 120.00 a = b = 99.75, c = 114.78, α = β = 90.00, γ = 120.00 a = b = 106.472, c = 71.323, α = β = 90, γ = 120 a = b = 158.101, c = 104.584, α = β = 90, γ = 120 a = b = 158.243, c = 104.657, α = β = 90, γ = 120 a = b = 157.866, c = 104.516, α = β = 90, γ = 120 a = b = 158.093, c = 104.072, α = β = 90, γ = 120 a = b = 157.914, c = 104.068, α = β = 90, γ = 120 a = b = 129.951, c = 67.809, α = β = γ = 90 a = b = 129.691, c = 67.905, α = β = γ = 90 a = b = 130.561, c = 68.160, α = β = γ = 90 CRYSTALLOGRAPHY REPORTS
I/sigma
91.0
4.4
48.538
98.2
4.8
58.095
98.8
4.9
53.602
95.5
6.6
35.159
91.1
6.7
15.786
97.3
4.5
20.306
98.2
6.3
46.182
96.4
5.1
49.325
98.5
10.6
12.602
99.0
8.3
18.790
97.0
5.7
41.443
99.5
5.2
59.295
99.9
14.1
14.892
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amino2,3dideoxythymidine (inhibitor 2)—were obtained. As was mentioned above, crystals were grown on earth parallel with the experiment performed on the ISS in the same conditions and using the same devices. First, the morphology of the earth and space grown crystals was visually inspected with a micro scope. In most cases the earth and spacegrown crys tals do not differ in morphology. The Xray diffraction data sets were collected from the spacegrown crystals and the best earthgrown crystals at the SPring 8 syn chrotron radiation facility at 100 K. The Xray data collections statistics are given in Table 2. All spacegrown crystals diffracted to higher reso lution than the earthgrown crystals. In some cases, for example, in the case of the complex of thymidine phosphorylase with 3amino2,3dideoxythymidine, crystals did not appear in capillaries that remained on earth, whereas the crystals grown in microgravity dif fracted to 1.85 Å resolution. All Xray data sets col lected from the spacegrown crystals have better char acteristics than the corresponding earthgrown crys tals and were suitable for determining the three dimensional structures of the corresponding proteins at high resolution. ACKNOWLEDGMENTS We thank our Japanese colleagues M. Sato, H. Tanaka, K. Inaka, and their coworkers for helpful advice, loading and assembling the crystallization box JCB, and assistance in collecting Xray diffraction data sets at the SPring 8 synchrotron radiation facility. This study was supported by the Central Scientific Research Institute of Mechanical Engineering of the Russian Federal Space Agency (Roscosmos) and the Russian Foundation for Basic Research, project no. 100901541.
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Translated by T. Safonova
