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1.  Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum  
Orotate phosphoribosyltransferase from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+.
Orotate phosphoribosyltransferase (OPRT) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with 5-α-d-phosphorylribose 1-diphosphate (PRPP) to yield diphosphate (PPi) and the nucleotide orotidine 5′-monophos­phate. OPRT from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+. The crystal exhibited tetragonal symmetry, belonging to space group P41 or P43, with unit-cell parameters a = b = 49.15, c = 226.94 Å. X-ray diffraction data were collected to 2.5 Å resolution at 100 K using a synchrotron-radiation source.
doi:10.1107/S1744309111043247
PMCID: PMC3274414  PMID: 22298010
orotate phosphoribosyltransferase; Plasmodium falciparum
2.  Crystallization and preliminary X-ray diffraction analysis of mouse prostaglandin F2α synthase, AKR1B3 
Aldo-keto reductase 1B3 (AKR1B3) produced in Escherichia coli has been crystallized in complex with NADPH by the sitting-drop vapour-diffusion method.
Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H2 (PGH2), which is a common intermediate of various prostanoids, to form PGF2α. AKR1B3 also reduces PGH2 to PGD2 in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P41212 or P43212, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source.
doi:10.1107/S1744309111036165
PMCID: PMC3232157  PMID: 22139184
aldo-keto reductases; AKR1B3; prostaglandin F2α synthase
3.  Crystallization and preliminary X-ray crystallographic analysis of a helicase-like domain from a tomato mosaic virus replication protein 
Crystals of the helicase domain from a tomato mosaic virus replication protein obtained using the hanging-drop vapour-diffusion method at 285 K diffracted X-rays to 2.05 Å resolution. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
doi:10.1107/S174430911104231X
PMCID: PMC3232162  PMID: 22139189
tomato mosaic virus; replicase protein; helicase domain
4.  Crystal Structure of the Superfamily 1 Helicase from Tomato Mosaic Virus 
Journal of Virology  2012;86(14):7565-7576.
The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.
doi:10.1128/JVI.00118-12
PMCID: PMC3416300  PMID: 22573863
5.  Three-dimensional, non-invasive, cross-sectional imaging of protein crystals using ultrahigh resolution optical coherence tomography 
Biomedical Optics Express  2012;3(4):735-740.
Micro-scale, non-invasive, three-dimensional cross-sectional imaging of protein crystals was successfully accomplished using ultra-high resolution optical coherence tomography (UHR-OCT) with low noise, Gaussian like supercontinuum. This technique facilitated visualization of protein crystals even those in medium that also contained substantial amounts of precipitates. We found the enhancement of the scattered signal from protein crystal by inclusion of agarose gel in the crystallization medium. Crystals of a protein and a salt in the same sample when visualized by UHR-OCT showed distinct physical characteristics, suggesting that protein and salt crystals may, in general, be distinguishable by UHR-OCT. UHR-OCT is a nondestructive and rapid method, which should therefore find use in automated systems designed to visualize crystals.
doi:10.1364/BOE.3.000735
PMCID: PMC3345802  PMID: 22574261
(110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging
6.  Structure of the Glycosyltransferase EryCIII in Complex with its Activating P450 Homologue EryCII 
Journal of Molecular Biology  2012;415-20(1):92-101.
In the biosynthesis of the clinically important antibiotic erythromycin D, the glycosyltransferase (GT) EryCIII, in concert with its partner EryCII, attaches a nucleotide-activated sugar to the macrolide scaffold with high specificity. To understand the role of EryCII, we have determined the crystal structure of the EryCIII·EryCII complex at 3.1 Å resolution. The structure reveals a heterotetramer with a distinctive, elongated quaternary organization. The EryCIII subunits form an extensive self-complementary dimer interface at the center of the complex, and the EryCII subunits lie on the periphery. EryCII binds in the vicinity of the putative macrolide binding site of EryCIII but does not make direct interactions with this site. Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT.
Graphical Abstract
Highlights
► First structure of macrolide GT and auxiliary protein. ► New system for the expression of EryCIII and EryCII. ► Auxiliary protein (EryCII) stabilizes GT (EryCIII). ► EryCII has cytochrome P450 fold but possesses an additional N-terminal helix.
doi:10.1016/j.jmb.2011.10.036
PMCID: PMC3391682  PMID: 22056329
GT, glycosyltransferase; LC-MS, liquid chromatography combined with mass spectrometry; Se-Met, selenomethionine; PDB, Protein Data Bank; glycosyltransferase; antibiotic synthesis; crystal structure; cytochrome P450
7.  Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis  
Crystals of the 45.1 kDa functional form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from B. subtilis diffracted to 2.30 Å resolution.
2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P21, with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S174430910804311X
PMCID: PMC2635871  PMID: 19194007
methionine-salvage pathway; Bacillus subtilis; RuBisCO; RuBisCO-like proteins; 2,3-diketo-5-methylthiopentyl-1-phosphate enolase
8.  Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger 
The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa has been crystallized by the sitting-drop vapour-diffusion method at 293 K.
The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca2+ to CBD1 results in conformational changes that stimulate exchange to exclude Ca2+ ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca2+-sensor. In order to clarify the structural scaffold for the Ca2+-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca2+-free form was attempted; the structure of the Ca2+-bound form is already available. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa was crystallized by the sitting-drop vapour-diffusion method at 293 K. The crystals belonged to the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 56.99, c = 153.86 Å, β = 120°, and contained one molecule per asymmetric unit (V M = 2.25 Å3 Da−1) with a solvent content of about 55% (V S = 45.57%). Diffraction data were collected within the resolution range 27.72–3.00 Å using an R-AXIS detector and gave a data set with an overall R merge of 10.8% and a completeness of 92.8%.
doi:10.1107/S1744309108032934
PMCID: PMC2593703  PMID: 19052365
Na+/Ca2+ exchanger; Ca2+-sensors
9.  Structure of the inhibitor complex of old yellow enzyme from Trypanosoma cruzi  
Journal of Synchrotron Radiation  2010;18(Pt 1):66-69.
The structures of old yellow enzyme from Trypanosoma cruzi which produces prostaglandin F2α from PGH2 have been determined in the presence or absence of menadione.
Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F2α from PGH2 have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF2α production specifically.
doi:10.1107/S0909049510033595
PMCID: PMC3004258  PMID: 21169695
X-ray structure; inhibitor complex; prostaglandin synthase
10.  Approach for growth of high-quality and large protein crystals 
Journal of Synchrotron Radiation  2010;18(Pt 1):16-19.
Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed.
Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein–precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.
doi:10.1107/S090904951003445X
PMCID: PMC3004246  PMID: 21169683
semi-solid agarose gels; top-seeded solution growth; large-scale hanging-drop method; X-ray crystallography; neutron crystallography
11.  Crystallization and preliminary neutron diffraction studies of HIV-1 protease cocrystallized with inhibitor KNI-272 
In order to determine the protonation states of the residues within the active site of an HIV-1 protease–inhibitor complex, a crystal of HIV-1 protease complexed with inhibitor (KNI-272) was grown to a size of 1.4 mm3 for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination.
This paper reports the crystallization and preliminary neutron diffraction measurements of HIV-1 protease, a potential target for anti-HIV therapy, complexed with an inhibitor (KNI-272). The aim of this neutron diffraction study is to obtain structural information about the H atoms and to determine the protonation states of the residues within the active site. The crystal was grown to a size of 1.4 mm3 by repeated macroseeding and a slow-cooling method using a two-liquid system. Neutron diffraction data were collected at room temperature using a BIX-4 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P21212, with unit-cell parameters a = 59.5, b = 87.4, c = 46.8 Å.
doi:10.1107/S1744309108029679
PMCID: PMC2581681  PMID: 18997326
HIV-1 protease; inhibitors; neutron diffraction
12.  Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment 
An RNA aptamer in complex with the human IgG Fc fragment have been crystallized. The stirring technique with a rotary shaker was used to improve the crystals and to ensure that they were of high quality and single, resulting in crystals that diffracted to 2.2 Å resolution.
Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution.
doi:10.1107/S1744309108028236
PMCID: PMC2564881  PMID: 18931441
RNA aptamers; Fc fragments; immunoglobulin G
13.  Conformational plasticity of RNA for target recognition as revealed by the 2.15 Å crystal structure of a human IgG–aptamer complex 
Nucleic Acids Research  2010;38(21):7822-7829.
Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.
doi:10.1093/nar/gkq615
PMCID: PMC2995045  PMID: 20675355
14.  Preparation, crystallization and preliminary crystallographic analysis of old yellow enzyme from Trypanosoma cruzi  
Old yellow enzyme from Trypanosoma cruzi, has been crystallized using the hanging-drop vapour-diffusion method.
Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F2α, a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 Å, β = 93.4° and two molecules per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 Å resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.
doi:10.1107/S1744309107044879
PMCID: PMC2339734  PMID: 17909300
old yellow enzyme; NADPH oxidoreductases
15.  Extracellular overproduction and preliminary crystallographic analysis of a family I.3 lipase 
A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed.
A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 Å resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P21, with unit-cell parameters a = 48.79, b = 84.06, c = 87.04 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.73 Å3 Da−1 and the solvent content was 55%.
doi:10.1107/S1744309107004575
PMCID: PMC2330184  PMID: 17329810
family I.3 lipases
16.  Crystallization and preliminary X-ray diffraction study of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis  
Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed.
Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed. Crystals were grown at 293 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.4 Å resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 Å. Assuming the presence of two molecules in the asymmetric unit, the V M value was 2.7 Å3 Da−1 and the solvent content was 54.1%. The protein was also cocrystallized with substrates and diffraction data were collected to 2.7 Å resolution.
doi:10.1107/S1744309107001388
PMCID: PMC2330122  PMID: 17277457
glycerol kinase; Thermococcus kodakaraensis; thermostability
17.  Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon 
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.
doi:10.1107/S1744309106030454
PMCID: PMC2242867  PMID: 16946475
pro-Tk-subtilisin; Thermococcus kodakaraensis
18.  Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum  
Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation.
Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å3 Da−1).
doi:10.1107/S1744309106015594
PMCID: PMC2243097  PMID: 16754976
orotidine 5′-monophosphate decarboxylase; Plasmodium falciparum
19.  Crystallization and preliminary X-ray analysis of the tRNA thiolation enzyme MnmA from Escherichia coli complexed with tRNAGlu  
The RNA thiouridylase MnmA in complex with tRNA was crystallized with and without ATP in three different crystal forms, which may reflect distinct sulfuration-reaction stages.
MnmA catalyzes a sulfuration reaction to synthesize 2-thiouridine at the wobble positions of tRNAGlu, tRNAGln and tRNALys in Escherichia coli. The binary complex of MnmA and tRNAGlu was crystallized in two different crystal forms: forms I and II. Cocrystallization of MnmA–tRNAGlu with ATP yielded form III crystals. The three crystal forms diffracted to 3.1, 3.4 and 3.4 Å resolution, respectively, using synchrotron radiation at SPring-8. These crystals belong to space groups C2, I212121 and C2, with unit-cell parameters a = 225.4, b = 175.8, c = 53.0 Å, β = 101.6°, a = 101.5, b = 108.0, c = 211.2 Å and a = 238.1, b = 102.1, c = 108.2 Å, β = 117.0°, respectively. The asymmetric units of these crystals are expected to contain two, one and two MnmA–tRNAGlu complexes, respectively.
doi:10.1107/S174430910600738X
PMCID: PMC2222564  PMID: 16582487
MnmA; tRNA; ATP; Escherichia coli
20.  Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus  
SecDF is a multi-path membrane protein required for efficient protein translocation and integration via translocon. Purification and crystallization of T. thermophilus SecDF have been achieved by exploiting unique crystallization techniques that allowed the collection of a 3.74 Å data set.
Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P43212. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.
doi:10.1107/S1744309106007779
PMCID: PMC2222563  PMID: 16582489
SecDF; membrane proteins; protein translocation; dehydration
21.  Crystallization and preliminary X-ray analysis of methylthioribose-1-phosphate isomerase from Bacillus subtilis  
Crystals of the 39 kDa functional form of methylthioribose-1-phosphate isomerase from B. subtilis diffracted to 2.50 Å.
Methylthioribose-1-phosphate isomerase (MtnA) from Bacillus subtilis, the first enzyme in the downstream section of the methionine-salvage pathway, was crystallized using the sitting-drop vapour-diffusion method. Crystals grew using ammonium sulfate as the precipitant at 293 K. They diffracted to 2.5 Å at 100 K using synchrotron radiation and were found to belong to the tetragonal space group P41, with unit-cell parameters a = b = 69.2, c = 154.7 Å. The asymmetric unit contains two molecules of MtnA, with a V M value of 2.4 Å3 Da−1 and a solvent content of 48%.
doi:10.1107/S1744309105015757
PMCID: PMC1952323  PMID: 16511105
methylthioribose-1-phosphate; methylthioribulose-1-phosphate; methylthioadenosine
22.  Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 
A human kynurenine aminotransferase II homologue from P. horikoshii OT3 has been overproduced in E. coli, purified, and characterized. Crystals of this protein have been obtained and analyzed by X-ray diffraction.
The Pyrococcus horikoshii OT3 genome contains a gene encoding a human kynurenine aminotransferase II (KAT II) homologue, which consists of 428 amino-acid residues and shows an amino-acid sequence identity of 30% to human KAT II. This gene was overexpressed in Escherichia coli and the recombinant protein (Ph-KAT II) was purified. Gel-filtration chromatography showed that Ph-KAT II exists as a homodimer. Ph-KAT II exhibited enzymatic activity that catalyzes the transamination of l-kynurenine to produce kynurenic acid. Crystals of Ph-KAT II were grown using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.2 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the centred orthorhombic space group C2221, with unit-cell parameters a = 71.75, b = 86.84, c = 137.30 Å. Assuming one molecule per asymmetric unit, the V M value was 2.19 Å3 Da−1 and the solvent content was 43.3%.
doi:10.1107/S1744309105005269
PMCID: PMC1952292  PMID: 16511030
kynurenine aminotransferase II; Pyrococcus horikoshii OT3
23.  Crystallization and preliminary X-ray diffraction study of thermostable RNase HIII from Bacillus stearothermophilus  
A thermostable ribonuclease HIII from B. stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K.
A thermostable ribonuclease HIII from Bacillus stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K. Native X-ray diffraction data were collected to 2.8 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 66.73, b = 108.62, c = 48.29 Å. Assuming one molecule per asymmetric unit, the V M value was 2.59 Å3 Da−1 and the solvent content was 52.2%.
doi:10.1107/S1744309105003659
PMCID: PMC1952286  PMID: 16511022
ribonuclease HIII
24.  Crystallization and preliminary X-ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 
The hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon A. pernix K1 was crystallized. The crystal diffracted to 2.7 Å resolution.
Thioredoxin peroxidase is a member of the peroxiredoxin family and plays a dominant role in a hydrogen peroxide metabolism. A recombinant form of the hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1, a polypeptide consisting of 250 amino acids, was purified. The C207S mutant protein was crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as the precipitant at 298 K. Diffraction data were collected and processed to 2.7 Å resolution. The crystal belongs to space group P1, with unit-cell parameters a = 126.2, b = 126.3, c = 213.7 Å, α = 80.4, β = 80.3, γ = 70.7°. Calculation of the self-rotation function showed that the protein quaternary structure includes a fivefold axis and five twofold axes.
doi:10.1107/S1744309105005294
PMCID: PMC1952277  PMID: 16511031
hyperthermostability; thioredoxin peroxidase; peroxiredoxins; archaea; Aeropyrum pernix K1

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