Preliminary Crystallographic Analysis Research Articles

Purification, crystallization and preliminary crystallographic analysis of Gan1D, a GH1 6-phospho-β-galactosidase from Geobacillus stearothermophilus T1

Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure-function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.

Cloning, purification, crystallization and preliminary X-ray studies of HMO2 from Saccharomyces cerevisiae.

The high-mobility group protein (HMO2) of Saccharomyces cerevisiae is a component of the chromatin-remodelling complex INO80, which is involved in double-strand break (DSB) repair. HMO2 can also bind DNA to protect it from exonucleolytic cleavage. Nevertheless, little structural information is available regarding these functions of HMO2. Since determination of three-dimensional structure is a powerful means to facilitate functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of HMO2 from S. cerevisiae are reported. The crystal belonged to space group P222, with unit-cell parameters a = 39.35, b = 75.69, c = 108.03 Å, and diffracted to a resolution of 3.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 3.19 Å(3) Da(-1).

Crystallization and preliminary crystallographic analysis of the chimeric protein LKB1-14-3-3ζ.

LKB1, a tumour suppressor, plays key roles in cell polarity, cell growth and energy metabolism. 14-3-3 proteins bind to LKB1 and suppress its functions. A chimera containing 14-3-3ζ and its binding region in LKB1 was constructed and the chimeric protein LKB1-14-4-3ζ was purified and crystallized. The crystal of LKB1-14-4-3ζ diffracted to 2.9 Å resolution and belonged to space group R32, with unit-cell parameters a = b = 130.262, c = 264.960 Å. Structure determination and refinement are in progress.

Purification, crystallization and preliminary crystallographic analysis of the ligand-binding regions of the PctA and PctB chemoreceptors from Pseudomonas aeruginosa in complex with amino acids.

Pseudomonas aeruginosa is an opportunistic pathogen and one of the major model organisms for the study of chemotaxis. The bacterium harbours 26 genes encoding chemoreceptors, most of which have not been annotated with a function. The paralogous chemoreceptors PctA and PctB (Pseudomonas chemotactic transducer A and B) were found to mediate chemotaxis towards L-amino acids. However, the ligand spectrum of the receptors is quite different since the recombinant ligand-binding region (LBR) of PctA binds 17 different L-amino acids whereas that of PctB recognizes only five. To determine the molecular basis underlying this ligand specificity, PctA-LBR and PctB-LBR have been purified and crystals have been produced after pre-incubation with L-Ile and L-Arg, respectively. Initial crystallization conditions have been identified by the counter-diffusion method and X-ray data have been collected at 2.5 Å (PctA-LBR bound to L-Ile) and 3.14 Å (PctB-LBR bound to L-Arg) resolution. Crystals belonged to space groups P2(1)2(1)2(1) and P3(1)2(1), with unit-cell parameters a = 72.2, b = 78.5, c = 116.6 Å and a = b = 111.6, c = 117.4, respectively, for PctA-LBR and PctB-LBR. Molecular-replacement methods will be pursued for structural determination.

Cloning, purification and preliminary crystallographic analysis of the complex of Helicobacter pylori α-carbonic anhydrase with acetazolamide.

Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori α-carbonic anhydrase (HpαCA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of HpαCA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å. An X-ray diffraction data set was collected from a single crystal to 1.7 Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an HpαCA dimer.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the putative NlpC/P60 endopeptidase, TTHA0266, from Thermus thermophilus HB8.

Autolysins belong to a protein family involved in peptidoglycan degradation and remodelling. Within this family, NlpC/P60 endopeptidases are involved in the hydrolysis of the peptide arm of peptidoglycan. In this work, the putative NlpC/P60 endopeptidase TTHA0266 from Thermus thermophilus HB8 was overexpressed, purified and crystallized. The crystals diffracted to 2.4 Å resolution and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 71.19, c = 198.68 Å, γ = 120°. Selenomethionine-substituted protein was crystallized and the structure was solved by single-wavelength anomalous dispersion.

Cloning, purification, crystallization and preliminary X-ray studies of the putative transcriptional regulator SPD0280 fromStreptococcus pneumoniaeD39

Streptococcus pneumoniae SPD0280 is a hypothetical protein that has been putatively identified as a transcriptional regulator. However, it has very low sequence identity to other well characterized transcriptional regulators. Determination of the three-dimensional structure may provide information for the characterization of proteins; therefore, it was decided to use X-ray diffraction analysis to learn more about this protein. Here, the expression, purification, crystallization and preliminary crystallographic analysis of SPD0280 from S. pneumoniae are reported. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.886, b = 66.785, c = 150.050 Å, and diffracted to a resolution of 2.5 Å. The crystals are likely to contain one molecule in the asymmetric unit, with a VM value of 2.06 Å(3) Da(-1).

Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a.

Type V myosins constitute the main cargo-transporting class of myosin motors in higher eukaryotes. They are mainly defined by their C-terminal globular domain, which is required for cargo binding as well as for motor auto-inhibition in the absence of cargo. To date, high-resolution structures only exist for globular domains from yeast. Since the majority of cellular cargoes in yeast are very different from the cargoes in higher eukaryotes, structural insights into the domain organization of globular domains from human type V myosins are important. The globular domain of human Myo5a was cloned, expressed and crystallized and data sets were collected. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.04, b = 86.70, c = 131.41 Å, α = β = γ = 90°, and diffracted with data-collection quality to 2.5 Å resolution.

Purification, crystallization and preliminary crystallographic analysis of the SpaA backbone-pilin subunit from probiotic Lactobacillus rhamnosus GG.

Lactobacillus rhamnosus GG, a widely used Gram-positive probiotic strain, is clinically well known for its perceived health-promoting effects. It has recently been shown to display proteinaceous pilus fibres (called SpaCBA) on its cell surface. Structurally, SpaCBA pili possess a characteristic three-pilin polymerized architecture, with repeating SpaA major pilins that form the backbone and two types of minor subunits (SpaB and SpaC). In this study, recombinant SpaA protein was purified, characterized and crystallized. The crystals diffracted to a resolution of 2.0 Å and belonged to space group C2, with unit-cell parameters a=227.9, b=63.2, c=104.3 Å, β=95.1°.

Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular β-galactosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a multi-enzyme system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo-acting extracellular enzymes that break down the high-molecular-weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure-function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P2₁2₁2₁, with average crystallographic unit-cell parameters of a=71.84, b=181.35, c=196.57 Å. Full diffraction data sets to 2.45 and 2.50 Å resolution have been collected for both the wild-type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash-cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three-dimensional crystal structure determination of GanB.

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori leucyl aminopeptidase-bestatin complex.

Helicobacter pylori is an important human pathogenic bacterium associated with numerous severe gastroduodenal diseases, including ulcers and gastric cancer. Cytosolic leucyl aminopeptidase (LAP) is an important housekeeping protein that is involved in peptide and protein turnover, catabolism of proteins and modulation of gene expression. LAP is upregulated in metronidazole-resistant H. pylori, which suggests that, in addition to having an important housekeeping role, LAP contributes to the mechanism of drug resistance. Crystals of H. pylori LAP have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belonged to the primitive triclinic space group P1, with unit-cell parameters a = 97.5, b = 100.2, c= 100.4 Å, α = 75.4, β = 60.9, γ = 81.8°. An X-ray diffraction data set was collected to 2.8 Å resolution from a single crystal. Molecular-replacement results using these data indicate that H. pylori LAP is a hexamer with 32 symmetry.

Purification, crystallization and preliminary crystallographic analysis of soybean mature glycinin A1bB2.

Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl₂, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate-citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6₃22, P2₁ and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, β = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, β = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined.

Crystallization and preliminary X-ray diffraction analysis of the α subdomain of Lon protease fromBrevibacillus thermoruber

DNA-binding ability has previously been reported as a novel function for the thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon), and the α subdomain (amino acids 491-605) of Bt-Lon has been identified as being responsible for DNA binding. However, the physiological role and DNA-recognition mode of Bt-Lon still remain unclear. In this study, the crystallization and preliminary crystallographic analysis of the Bt-Lon α subdomain are presented. Native diffraction data to 2.88 Å resolution were obtained from a vitrified crystal at 100 K on the BL13C1 beamline at the NSRRC (National Synchrotron Radiation Research Center), Taiwan. The crystals belonged to space group P23, with unit-cell parameters a = b = c = 94.28 Å. Solvent-content calculations and molecular-replacement results suggest that there are two molecules of Bt-Lon α subdomain per asymmetric unit.

Preliminary crystallographic analysis of neuraminidase N2 from a new influenza A virus

Influenza virus is a major viral respiratory pathogen that causes yearly epidemics in temperate climates. The H3N2 subtype is one of the major causative agents of severe epidemics and plays a critical role in vaccine development. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are two commercially available NA-targeted competitive antiviral drugs. However, their effectiveness has been compromised by the rapid emergence of resistance. Q136K is a novel mutation in NA which confers resistance to zanamivir. In this study, a Q136K mutant N2 protein was expressed in a baculovirus system and crystals were obtained. The crystal of N2 belonged to space group P2₁2₁2₁, with unit-cell parameters a = 109.5, b = 112.8, c = 165.2 Å. Data were collected to 2.4 Å resolution. Four monomers were found in the asymmetric unit. The Matthews coefficient and solvent content were calculated to be 3.0 ų Da⁻¹ and 59.0%, respectively.

Expression, purification, crystallization and preliminary crystallographic analysis of spermidine acetyltransferase from Escherichia coli.

The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.

Expression, purification, crystallization and preliminary crystallographic analysis of hepatitis B virus core protein dimerized via a peptide linker containing an EGFP insertion.

Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.

Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies

Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.

Purification, crystallization and preliminary X-ray crystallographic study of the tRNA pseudouridine synthase TruB from Streptococcus pneumoniae.

The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine (Ψ) at residue 55 of elongator tRNAs. In order to better elucidate the functions of TruB in the formation of pseudouridine, the three-dimensional structure of full-length TruB was determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported. The crystal belonged to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 Å, β = 102.05°, and diffracted to a resolution of 1.7 Å. The crystal is most likely to contain one molecule in the asymmetric unit, with a VM value of 2.40 Å(3) Da(-1).

Crystallization and preliminary crystallographic studies of GRASP65 GRASP domain from Rattus norvegicus.

GRASP65 and GRASP55 were classified as Golgi reassembly stacking proteins which play crucial and complementary roles in the stacking of Golgi cisternae. They also participate in vesicle tethering, mitotic progression, the disassembly and reassembly of the Golgi apparatus during mitosis and unconventional secretory pathway regulation. In this study, the expression, crystallization and preliminary crystallographic analysis of the GRASP65 GRASP domain from Rattus norvegicus are presented. The crystals diffracted to 2.0 Å resolution and belonged to space group P21212, with unit-cell parameters a = 44.99, b = 104.29, c = 37.93 Å, α = β = γ = 90°. Furthermore, molecular replacement was employed to determine the structure of the GRASP65 GRASP domain from R. norvegicus.

Expression, crystallization and preliminary crystallographic study of GluB from Corynebacterium glutamicum.

GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 82.50, c = 72.69 Å.