The flavoenzyme fructosyl peptide oxidase (FPOX) catalyses the oxidative deglycation of fructosyl amino acids or fructosyl dipeptides to produce amino acids, glucosone and hydrogen peroxide. In this study, FPOX protein from Eupenicillium terrenum sp. (EtFPOX) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. EtFPOX crystals were obtained using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 65.6, b = 80.0, c = 83.4 Å, and contained one molecule in the asymmetric unit. The calculated Matthews coefficient and solvent content were 2.22 Å(3) Da(-1) and 44.62%, respectively.
Peptide release factor 1 (RF1) regulates the termination of translation in protein synthesis by recognizing the stop codons. The eukaryotic RF1s (eRF1s) from Arabidopsis thaliana and human have different stop-codon preferences even though they share high sequence similarity. Based on known RF1 structures, it has been suggested that the specificity depends on both the local structure and the domain arrangement, but the lack of a structure of Arabidopsis eRF1 hinders a detailed comparison. To reveal the mechanism of stop-codon recognition and compare it with that of human eRF1, one of the three Arabidopsis eRF1s, AteRF1-1, was studied and a preliminary X-ray crystallographic analysis is reported here. The protein was overexpressed in Escherichia coli and crystallized at room temperature using the vapour-diffusion method. Crystals were grown from 1.6 M lithium sulfate, 0.1 M Tris-HCl pH 8.0, 2%(v/v) PEG 400 and diffracted to 3.77 Å resolution. The data were processed in point group 622, with unit-cell parameters a = b = 136.6, c = 325.7 Å.
Abscisic acid (ABA) modulates many complicated developmental processes and responses to environmental stimuli. Recently, several (+)-ABA signalling mechanisms by the RCAR/PYR1/PYL family of proteins (PYLs) have been proposed. However, the mechanism of the recognition and binding of the unnatural ligand (-)-ABA by PYLs has not yet been elucidated. In the present study, the expression, purification and crystallization of PYL3 in complex with (-)-ABA are reported. Diffraction data were refined to 2.65 Å resolution for this complex in space group P65. These findings will help to explain the stereospecificity of PYLs for (-)-ABA and to explore the selective ABA agonists.
N(6)-methyladenosine (m6A) is a ubiquitous modification found in mammalian mRNA and long noncoding RNA. ALKBH5 is a member of the iron(II)- and 2-oxoglutarate-dependent AlkB oxygenase family and has been shown to catalyze the oxidative demethylation of N(6)-methyladenosine in RNA. The ALKBH5 protein was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.456, b = 83.406, c = 92.909 Å, α = β = γ = 90.00° and one molecule in the asymmetric unit.
Improving the specificity of α-cyclodextrin glucanotransferase is a significant issue in the field of α-cyclodextrin production. In this study, a constructed Y167H mutant α-cyclodextrin glucanotransferase with enhanced α-cyclodextrin specificity was successfully expressed and purified. Single crystals were grown using PEG 4000 as a precipitating agent by the hanging-drop vapour-diffusion method at 293 K. The crystals exhibited two kinds of morphology in different crystallization conditions. The crystals diffracted to at least 2.2 Å resolution (space group P2₁2₁2₁), with unit-cell parameters a=65.69, b=78.70, c=137.00 Å. Assuming the asymmetric cell to be occupied by a monomer of 75 kDa, the unit cell contains 43.77% solvent with a crystal volume per protein mass, VM, of 2.19 Å3 Da(-1).
Peptidylarginine deiminase (PAD) catalyzes the post-translational conversion of peptidylarginine to peptidylcitrulline in the presence of calcium ions. Among the five known human PAD isozymes (PAD1-4 and PAD6), PAD1 exhibits the broadest substrate specificity. Crystals of PAD1 obtained using polyethylene glycol 3350 as a precipitant diffracted to 3.70 Å resolution using synchrotron radiation. Two PAD1 molecules were contained in the asymmetric unit and the crystals belonged to space group P6(1), with unit-cell parameters a = b = 90.3, c = 372.3 Å. The solvent content was 58.2%.
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.
Long-term nonprogression during simian immunodeficiency virus (SIV) infection has been strongly associated with the major histocompatibility complex (MHC) class I allele Mamu-B*17. Here, a complex of rhesus macaque Mamu-B*17 with rhesus macaque β2-microglobulin (β2m) and an immunodominant peptide (SIVmac239 Env241-251; LRCNDTNYSGF; Env LF11) derived from the SIV Env protein was crystallized by the hanging-drop method using PEG 3350 as a precipitating agent. The crystals belonged to the primitive monoclinic space group P2, with unit-cell parameters a = 68.3, b = 45.0, c = 81.5 Å, β = 96.5°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.96 Å(3) Da(-1) and 58.5%, respectively.
Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell parameters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.
Wuhan nodavirus (WhNV), which is a new member of the Nodaviridae family, encodes a viral protein, B2, that suppresses RNA silencing and host-cell RNA interference (RNAi)-mediated immunity. Although Flock House virus (FHV), another member of the Nodaviridae family, also produces a B2 protein with a similar function, the primary sequences of the B2 proteins from WhNV and FHV have no similarity. To gain a better understanding of the structural details and the mechanism of suppression of RNA silencing by WhNV B2 and to compare it with FHV B2, recombinant WhNV B2 protein has been overexpressed in Escherichia coli, purified and crystallized at 291 K using PEG 4000 as a precipitant. A 2.8 Å resolution data set has been collected from a single crystal at 100 K. This crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a=27.3, b=45.6, c=133.9 Å, α=β=γ=90°. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient is 2.2 Å3 Da(-1).
Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to inject effector proteins into rival cells in niche competition. Tse3, one of the effectors of T6SS, is delivered into the periplasm of recipient cells. Tse3 functions as a muramidase that degrades the β-1,4-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan, thus leading to lysis of the recipient cells and providing a competitive advantage to the donor cells. Here, the preliminary crystallographic study of Tse3 is reported. A crystal of Tse3 diffracted to 1.5 Å resolution. It belonged to space group C121, with unit-cell parameters a = 166.99, b = 70.13, c = 41.94 Å, α = 90.00, β = 90.52, γ = 90.00° and one molecule per asymmetric unit.
Integrin cytoplasmic domain-associated protein-1 (ICAP1) is a suppressor of integrin activation and directly binds to the cytoplasmic tail of β1 integrins; its binding suppresses integrin activation by competition with talin. Krev/Rap1 interaction trapped-1 (KRIT1) releases ICAP1 suppression of integrin activation by sequestering ICAP1 away from integrin cytoplasmic tails. Here, the cocrystal structure of the PTB domain of ICAP1 in complex with a 29-amino-acid fragment (residues 170-198) of KRIT1 is presented to 1.7 Å resolution [the resolution at which 〈I/σ(I)〉 = 2.9 was 1.83 Å]. In previous studies, the structure of ICAP1 with integrin β1 was determined to 3.0 Å resolution and that of ICAP1 with the N-terminal portion of KRIT1 (residues 1-198) was determined to 2.54 Å resolution; therefore, this study provides the highest resolution structure yet of ICAP1 and allows further detailed analysis of the interaction of ICAP1 with its minimal binding region in KRIT1.
Dibenzothiophene (DBT) and its derivatives are typical sulfur compounds found in fossil fuels. These compounds show resistance to the hydrodesulfurization treatment that is commonly used in industry. Dibenzothiophene monooxygenase (DszC) is responsible for the oxidation of DBT, which is the first and the rate-limiting step in the DBT enzymatic desulfurization 4S pathway. In this study, the crystal structure of DszC from Rhodococcus erythropolis DS-3 is reported. The crystal of native DszC belonged to space group P1, with unit-cell parameters a = 96.16, b = 96.27, c = 98.56 Å, α = 81.03, β = 67.57, γ = 85.84°. To determine the phase, SAD X-ray diffraction data were collected from a SeMet-derivative DszC crystal, which also belonged to space group P1, with unit-cell parameters a = 95.379, b = 95.167, c = 94.891 Å, α = 87.046, β = 70.536, γ = 79.738°. Further structural analysis of DszC is in progress.
The Escherichia coli cyclic AMP receptor protein (CRP) is a prokaryotic global transcription activator protein that controls the expression of many different genes. Wild-type CRP can bind to special DNA sequences in the presence of cAMP. The substitution of Asp53 by His results in the CRP* phenotype, which does not require exogenous cAMP. In the present study, the D53H CRP mutant was overexpressed, purified and crystallized. cAMP-free D53H CRP crystals were obtained and diffracted to a resolution of 2.9 Å. Based on the systematic absences of the crystals, the space group is likely to be P2(1)2(1)2(1), with unit-cell parameters a = 76.66, b = 152.14, c = 176.11 Å. The asymmetric unit was confirmed to contain four protein dimers, with a Matthews coefficient of 2.71 Å(3) Da(-1) and a solvent content of 54.68%.
DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I2₁2₁2₁ and the diffraction resolution is 3.0 Å with unit-cell parameters a=47.68, b=104.45, c=234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a VM value of 3.24 Å3 Da(-1) and a solvent content of 62.1%.
The Escherichia coli cyclic AMP receptor protein (CRP) is a well known transcription activator protein. In this study, CRP was overexpressed, purified and cocrystallized with cAMP and a 38 bp full-length double-stranded DNA fragment. The full-length segment differed from the half-site fragments used in previous crystallization experiments and is more similar to the environment in vivo. CRP-cAMP-DNA crystals were obtained and diffracted to 2.9 Å resolution. The crystals belonged to space group P3121, with unit-cell parameters a = b = 76.03, c = 144.00 Å. The asymmetric unit was found to contain one protein molecule and half a 38 bp full-length double-stranded DNA fragment, with a Matthews coefficient of 2.62 Å(3) Da(-1) and a solvent content of 53.14%.
MoHrip2, a novel effector protein from the pathogenic fungus Magnaporthe oryzae, was purified and crystallized using the sitting-drop vapour-diffusion method. Native crystals and selenomethionine-labelled crystals were obtained using 2.2 M ammonium sulfate as a precipitant. A native data set was collected to 2.0 Å resolution at 100 K using an in-house X-ray source and a selenomethionine-labelled data set containing anomalous signal was collected to 1.8 Å resolution at 100 K using a synchrotron source. Based on the anomalous signal generated from the Se atom, the MoHrip2 structure was successfully solved using the single-wavelength anomalous dispersion (SAD) method.
The novel thermostable esterase EstL5 belonging to the GDSL family exhibits a unique cold-adaptation feature at low temperatures. To better understand its biochemical and enzymatic properties, recombinant EstL5 protein was purified and crystallized using the vapour-diffusion method. The EstL5 crystals diffracted X-rays to 2.79 Å resolution using a synchrotron-radiation source, belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 101.51, c = 124.22 Å, and are expected to contain two molecules in each asymmetric unit. To obtain initial phases, selenomethionyl-substituted protein was overproduced. Purified SeMet-labelled EstL5 protein was crystallized and formed crystals that diffracted to a resolution of 3.0 Å.
Alanine dehydrogenase (OF4Ald) from the alkaliphilic Bacillus pseudofirmus OF4 was expressed and purified with a His6 tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K using a solution consisting of 0.1 M Tris-HCl pH 8.0, 0.2 M LiSO4, 22%(w/v) PEG 3350. X-ray diffraction data were collected to 2.8 Å resolution. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 88.04, b = 105.59, c = 120.53 Å, α = 88.37, β = 78.77, γ = 82.65°.
Arabidopsis thaliana (At) MAP4Kalpha2, a member of the Ste20/PAK-like protein kinase family, is an essential component of the septum initiation network involved in cell division. To better understand the mode of action of AtMAP4Kalpha2, a structural biology approach has been pursued. In this study, the kinase domain of AtMAP4Kalpha2 was cloned, expressed, purified and crystallized. The crystals diffracted to 1.9 Å resolution and belonged to space group C2221, with unit-cell parameters a = 55.27, b = 82.93, c = 133.15 Å.
