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1.  Ellman’s reagent in promoting crystallization and structure determination of Anabaena CcbP 
Acta Crystallographica Section F  2012;68(Pt 11):1409-1414.
Ellman’s reagent oxidized the free sulfhydryl group of cysteine in Anabaena CcbP protein, facilitating its crystallization.
Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman’s reagent [5,5′-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that DTNB modification facilitated crystallization of CcbP by inducing polar inter­actions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.
doi:10.1107/S1744309112034938
PMCID: PMC3515393  PMID: 23143261
Ellman’s reagent; chemical modification; Anabaena CcbP
2.  High-resolution structure of a new crystal form of BamA POTRA4–5 from Escherichia coli  
The crystal structure of POTRA4–5 has been determined to 1.50 Å resolution with an R factor of 14.7% and an R free of 18.9%.
In Escherichia coli, the BAM complex is employed to mediate correct folding of the outer membrane (OM) proteins into β-barrels and their insertion into the OM. BamA, which is an essential component of the complex, consists of a C-­terminal transmembrane region and five N-terminal polypeptide transport-associated (POTRA) domains. Although deletion studies have shown that each of the POTRA domains plays an important role in the process of BAM complex formation, only POTRA5 is essential for cell viability. Here, the crystal structure of POTRA4–5 has been determined to 1.50 Å resolution with an R factor of 14.7% and an R free of 18.9%.
doi:10.1107/S1744309111014254
PMCID: PMC3144785  PMID: 21795783
BAM complex; POTRA domains; BamA; Escherichia coli
3.  Preliminary X-ray crystallographic studies of the catalytic subunit of Escherichia coli AHAS II with its cofactors 
In this study, the catalytic subunit of E. coli AHAS II was cocrystallized with its cofactors Mg2+, FAD and ThDP using the sitting-drop vapour-diffusion method and the crystals diffracted to 2.80 Å resolution.
Acetohydroxyacid synthase (AHAS) is the first common enzyme in the branched-chain amino-acid biosynthesis pathway and is the target of several classes of commercial herbicides. In this study, the Escherichia coli ilvG gene that encodes the catalytic subunit of AHAS II was cloned into the pET28a vector and expressed in soluble form at high levels in E. coli strain BL21 (DE3) cells. The protein was purified using Ni2+-chelating chromatography followed by size-exclusion chromatography. The catalytic subunit of E. coli AHAS II was cocrystallized with its cofactors Mg2+, FAD and ThDP using the sitting-drop vapour-diffusion method and the crystals diffracted to 2.80 Å resolution.
doi:10.1107/S1744309111008839
PMCID: PMC3107135  PMID: 21636904
acetohydroxyacid synthase; Escherichia coli; AHAS II; branched-chain amino-acid synthesis
4.  Structure of the putative dihydroorotate dehydrogenase from Streptococcus mutans  
The crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Å resolution.
Streptococcus mutans is one of the pathogenic species involved in dental caries, especially in the initiation and development stages. Here, the crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Å resolution. DHOD is a flavin mononucleotide-containing enzyme which catalyzes the oxidation of l-dihydroorotate to orotate, which is the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. The reductive lysine-methylation procedure was applied in order to improve the diffraction qualities of the crystals. Analysis of the S. mutans DHOD crystal structure shows that this enzyme is a class 1A DHOD and also suggests potential sites that could be exploited for the design of highly specific inhibitors using the structure-based chemotherapeutic design technique.
doi:10.1107/S1744309110048414
PMCID: PMC3034605  PMID: 21301083
dihydroorotate dehydrogenases; Streptococcus mutans; pyrimidine biosynthesis
5.  Purification, crystallization and preliminary X-ray crystallographic analysis of 23S RNA m2G2445 methyltransferase RlmL from Escherichia coli  
RlmL, a 23S rRNA m2G2445 methyltransferase from Escherichia coli, was expressed, purified and crystallized, and crystals diffracted to 2.2 Å.
The RlmL (YcbY) protein in Escherichia coli is an rRNA methyltransferase that is specific for m2G2445 modification of 23S RNA. The rlmL gene was cloned into the expression vector pET28a and expressed in the host E. coli strain BL21 (DE3). Recombinant protein with a six-histidine tag was purified by Ni2+-affinity chromatography followed by gel filtration. Crystals were grown using the hanging-drop vapour-diffusion method and a detergent was used as an additive to improve diffraction quality. The final crystals diffracted to 2.2 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 73.6, b = 140.8, c = 102.9 Å, β = 102.3°. The crystal has a most probable solvent content of 62.8% with two molecules in the asymmetric unit.
doi:10.1107/S1744309110035074
PMCID: PMC3001654  PMID: 21045301
RlmL; rRNA methyltransferases; Echerichia coli
6.  The structure of the hypothetical protein smu.1377c from Streptococcus mutans suggests a role in tRNA modification 
The crystal structure of smu.1377c, a hypothetical protein from S. mutans, shows a similar fold to Sua5_YciO_YrdC-family proteins and indicates its functional role in tRNA modification.
Members of the Sua5_YciO_YrdC protein family are found in both eukaryotes and prokaryotes and possess a conserved α/β twisted open-sheet fold. The Escherichia coli protein YrdC has been shown to be involved in modification of tRNA. The crystal structure of smu.1377c, a hypothetical protein from Streptococcus mutans, has been determined to 2.25 Å resolution. From structure analysis and comparison, it is shown that smu.1377c is a member of the Sua5_YciO_YrdC family and that it may play the same role as E. coli YrdC.
doi:10.1107/S1744309110018944
PMCID: PMC2898458  PMID: 20606270
smu.1377c; PF01300; tRNA modification; Streptococcus mutans
7.  Structure of orotate phosphoribosyltransferase from the caries pathogen Streptococcus mutans  
The crystal structure of OPRTase from the caries pathogen Streptococcus mutans is reported at 2.4 Å resolution.
Orotate phosphoribosyltransferase (OPRTase) catalyzes the OMP-forming step in de novo pyrimidine-nucleotide biosynthesis. Here, the crystal structure of OPRTase from the caries pathogen Streptococcus mutans is reported at 2.4 Å resolution. S. mutans OPRTase forms a symmetric dimer and each monomer binds two sulfates at the active sites. The structural symmetry of the sulfate-binding sites and the missing loops in this structure are consistent with a symmetric catalysis mechanism.
doi:10.1107/S1744309110009243
PMCID: PMC2864676  PMID: 20445243
orotate phosphoribosyltransferase; Streptococcus mutans
8.  Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutans  
Purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, has been expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique.
The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and α-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 Å.
doi:10.1107/S1744309109045059
PMCID: PMC2802883  PMID: 20054131
purine nucleoside phosphorylase; Streptococcus mutans; nucleotide-salvage pathway
9.  Preliminary X-ray crystallographic analysis of a nitric oxide-inducible lactate dehydrogenase from Staphylococcus aureus  
A nitric oxide-inducible lactate dehydrogenase from S. aureus, Sa-LDH-1, has been cloned, expressed, purified and crystallized in space group C2, with unit-cell parameters a = 131.4, b = 74.4, c = 103.2 Å, β = 133.4°.
Recent studies have indicated that Staphylococcus aureus can survive the nitrosative stress (caused by the radical nitric oxide; NO·) mounted by the immune system of the infected host. It does this by expressing a nitric oxide-inducible l-lactate dehydrogenase (Sa-LDH-1). Therefore, if efficient inhibitors of Sa-LDH-1 can be designed then Sa-LDH-1 could be a potential drug target against the pathogen S. aureus. For this purpose, the nitric acid-inducible LDH-1 from S. aureus COL strain has been cloned into the expression vector pET-28a(+) and the protein has been expressed, purified and crystallized. The Sa-­LDH-1 crystal diffracted to 2.4 Å resolution at a home X-ray source and belonged to space group C2, with unit-cell parameters a = 131.4, b = 74.4, c = 103.2 Å, β = 133.4°.
doi:10.1107/S1744309109036173
PMCID: PMC2765899  PMID: 19851020
nitric oxide-inducible lactate dehydrogenase; Staphylococcus aureus
10.  Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteria 
All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work.
All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. Two dUTPase-encoding genes, yncF and yosS, have been identified in Bacillus subtilis. The gene dut, encoding dUTPase from the dental pathogen Streptococcus mutans, was amplified from S. mutans genomic DNA. The three genes were cloned into expression vectors and overexpressed at high levels in Escherichia coli. Each protein was purified in two steps using chromatographic methods. Crystals of the YosS and YncF proteins and of S. mutans dUTPase were obtained using the vapour-diffusion method. X-ray diffraction data sets were collected from crystals of selenomethionine-labelled YosS and S. mutans dUTPase to resolutions of 2.3 and 1.7 Å, respectively. The crystal of native YncF diffracted to 2.7 Å resolution.
doi:10.1107/S1744309109006228
PMCID: PMC2664754  PMID: 19342774
dUTPases; Streptococcus mutans; Bacillus subtilis
11.  Crystallization and preliminary X-ray crystallographic analysis of SMU.412c protein from the caries pathogen Streptococcus mutans  
Crystallization of SMU.412c protein from the caries pathogen Streptococcus mutans can easily appear in the condition 2.8 M sodium acetate pH 7.0 and its crystal belongs to space group P41212.
The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His6 tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8 Å resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P41212, with unit-cell parameters a = b = 53.5, c = 141.1 Å.
doi:10.1107/S1744309109009464
PMCID: PMC2664769  PMID: 19342789
SMU.412c; dental caries; Streptococcus mutans; histidine triad-like proteins
12.  Preliminary X-ray crystallographic studies of Bacillus subtilis SpeA protein 
In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme the speA gene was amplified from B. subtilis genomic DNA and cloned. The enzyme was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 298 K.
The speA gene in Bacillus subtilis encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine. Arginine decarboxylase is an important enzyme in polyamine metabolism in B. subtilis. In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme, the speA gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET-28a(+). SpeA was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 289 K. The best crystal diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 86.4, b = 63.3 c = 103.3 Å, β = 113.9°.
doi:10.1107/S1744309109003856
PMCID: PMC2650450  PMID: 19255484
SpeA; Bacillus subtilis; arginine decarboxylases
13.  Crystallization and preliminary X-ray analysis of human liver α-enolase 
α-Enolase from human liver (hENO1) was expressed as a soluble protein and purified by affinity column chromatography and gel filtration. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.5 Å resolution.
Enolase is a multifunctional enzyme that plays important roles in many biological and disease processes. α-Enolase from human liver (hENO1) was expressed as a soluble protein and purified by affinity column chromatography and gel filtration. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.5 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 72.85, b = 66.02, c = 79.43 Å, β = 94.54°.
doi:10.1107/S1744309109004138
PMCID: PMC2650463  PMID: 19255486
human liver α-enolase
14.  Preliminary X-ray crystallographic analysis of SMU.573, a putative sugar kinase from Streptococcus mutans  
SMU.573 from S. mutans was expressed in E. coli and crystallized. The crystals belong to space group I4 and 2.5 Å resolution diffraction data were collected at an in-house chromium radiation source.
SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni2+-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 Å resolution diffraction data set was collected using an in-house chromium radiation source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 Å, α = β = γ = 90°.
doi:10.1107/S1744309107065645
PMCID: PMC2373987  PMID: 18097102
SMU.573; Streptococcus mutans
15.  Protein preparation, crystallization and preliminary X-ray analysis of the C-terminal domain of human RSK1 serine/threonine protein kinase 
The C-terminal catalytic domain of serine/threonine kinase RSK1 has been crystallized; the crystals diffracted to a resolution of 2.7 Å and belonged to space group P21.
As a substrate of extracellular signal-related kinase (ERK), the p90 ribosome S6 kinase 1 (RSK1) is at the terminus of the Ras/ERK pathway. Residues 411–735 of human RSK1, covering the C-terminal serine/threonine kinase catalytic domain and the functionally important tail, were cloned into an Escherichia coli expression vector. The protein was expressed, purified and crystallized. The crystals diffracted to 2.7 Å and belonged to space group P21, with unit-cell parameters a = 39.8, b = 143.8, c = 59.9 Å, β = 95.7°.
doi:10.1107/S1744309107051329
PMCID: PMC2344107  PMID: 18084084
p90 ribosome S6 kinase 1; serine/threonine kinases
16.  Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans  
The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2.
The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.
doi:10.1107/S1744309107043242
PMCID: PMC2339730  PMID: 17909295
SMU.961; Streptococcus mutans
17.  Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants  
A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution.
The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-­phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P212121, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.
doi:10.1107/S1744309107040304
PMCID: PMC2376312  PMID: 17768362
SMU.636; Streptococcus mutans;  glucosamine 6-­phosphate deaminase
18.  Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans  
A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution.
d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU_599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3121 or P3221, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.
doi:10.1107/S1744309107040298
PMCID: PMC2376309  PMID: 17768361
d-alanine-d-alanine ligase; drug design; Streptococcus mutans
19.  Purification, crystallization and preliminary X-ray analysis of the glucosamine-6-phosphate N-acetyltransferase from human liver 
Glucosamine-6-phosphate N-acetyltransferase from human liver was expressed, purified and crystallized. Diffraction data have been collected to 2.6 Å resolution.
Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of d-glucosamine 6-phosphate to form N-acetyl-d-glucosamine 6-­phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni2+-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 Å resolution. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 50.08, c = 142.88 Å.
doi:10.1107/S1744309106037730
PMCID: PMC2225216  PMID: 17077487
glucosamine-6-phosphate N-acetyltransferase
20.  Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans  
Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution.
The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni2+-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P21, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.
doi:10.1107/S1744309106035640
PMCID: PMC2225190  PMID: 17012790
methionine synthase; Streptococcus mutans
21.  Preparation, crystallization and preliminary X-ray analysis of protein YtlP from Bacillus subtilis  
The crystallization and preliminary X-ray crystallographic analysis of protein YtlP from B. subtilis is reported.
Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2′-5′ RNA-ligase family. It contains 183 residues and two copies of the HXTX sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2′-5′ RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified to homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 Å and belonged to space group P212121, with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 Å.
doi:10.1107/S174430910603199X
PMCID: PMC2225199  PMID: 17012785
YtlP; 2′-5′ RNA-ligase family; Bacillus subtilis
22.  Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis  
B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution.
Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°.
doi:10.1107/S1744309105011012
PMCID: PMC1952297  PMID: 16511078
YjcG

Results 1-22 (22)