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1.  Structure of an Escherichia coli N-acetyl-d-neuraminic acid lyase mutant, E192N, in complex with pyruvate at 1.45 Å resolution 
Acta Crystallographica Section F  2009;65(Pt 11):1088-1090.
The 1.45 Å structure of E. coli N-acetyl-d-neuraminic acid lyase in complex with pyruvate in space group P212121 is reported from new low-salt crystallization conditions that will facilitate soaking experiments with substrates and inhibitors.
The structure of a mutant variant of Escherichia coli N-acetyl-d-neuraminic acid lyase (NAL), E192N, in complex with pyruvate has been determined in a new crystal form. It crystallized in space group P212121, with unit-cell parameters a = 78.3, b = 108.5, c = 148.3 Å. Pyruvate has been trapped in the active site as a Schiff base with the catalytic lysine (Lys165) without the need for reduction. Unlike the previously published crystallization conditions for the wild-type enzyme, in which a mother-liquor-derived sulfate ion is strongly bound in the catalytic pocket, the low-salt conditions described here will facilitate the determination of further E. coli NAL structures in complex with other active-site ligands.
doi:10.1107/S1744309109037403
PMCID: PMC2777032  PMID: 19923724
N-acetyl-d-neuraminic acid lyase; directed evolution; Schiff base; aldolases
2.  Crystallization and crystallographic analysis of Bacillus subtilis xylanase C 
Acta Crystallographica Section F  2009;65(Pt 5):499-503.
XynC, a glycosyl hydrolase family 5 xylanase from B. subtilis, has been purified and crystallized with the primary goal of structural determination and characterization of a second xylanase from this large catalytically diverse glycosyl hydrolase family.
The recent biochemical characterization of the xylanases of glycosyl hydrolase family 5 (GH 5) has identified a distinctive endo mode of action, hydrolyzing the β-1,4 xylan chain at a specific site directed by the position of an α-1,2-linked glucuronate moiety. Xylanase C (XynC), the GH 5 xylanase from Bacillus subtilis 168, has been cloned, overexpressed and crystallized. Initial data collection was performed and a preliminary model has been built into a low-quality 2.7 Å resolution density map. The crystals belonged to the primitive monoclinic space group P21. Further screening identified an additive that resulted in large reproducible crystals. This larger more robust crystal form belonged to space group P21212 and a resulting data set has been processed to 1.64 Å resolution. This will be the second structure to be solved from this unique xylanase family and the first from a Gram-positive bacterium. This work may help to identify the structural determinants that allow the exceptional specificity of this enzyme and the role it plays in the biological depolymerization and processing of glucuronoxylan.
doi:10.1107/S1744309109013098
PMCID: PMC2675595  PMID: 19407387
xylanase C; Bacillus subtilis; family 5 xylanases
3.  Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB 
The major outer membrane protein PorB from N. meningitidis was crystallized in three crystal forms; the best X-ray diffraction data were collected to 2.3 Å resolution.
The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P63 crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.
doi:10.1107/S1744309109032333
PMCID: PMC2765884  PMID: 19851005
outer membrane proteins; Neisseria meningitidis; denaturation; refolding; detergents; β-barrel membrane proteins; porins
4.  Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB 
The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit while both the R32 and P63 crystal forms contain one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.
doi:10.1107/S1744309109032333
PMCID: PMC2765884  PMID: 19851005
outer membrane protein; Neisseria meningitides; denaturation; refolding; detergent; β-barrel membrane protein; porin
5.  Role of tyrosine 131 in the active site of paAzoR1, an azoreductase with specificity for the inflammatory bowel disease prodrug balsalazide 
Tyr131 plays an important role in determining the substrate specificity and thermal stability of azoreductase 1 from P. aeruginosa (pAzoR1). The structure shows that the substrate methyl red binds deeper in the active site of the mutant, which is reflected in the change in enzymic properties.
Azoreductase 1 from Pseudomonas aeruginosa strain PAO1 (paAzoR1) catalyses the activation of the prodrug balsalazide and reduces the azo dye methyl red using reduced nicotinamide adenine dinucleotide cofactor as an electron donor. To investigate the mechanism of the enzyme, a Y131F mutation was introduced and the enzymic properties of the mutant were compared with those of the wild-type enzyme. The crystallographic structure of the mutant with methyl red bound was solved at 2.1 Å resolution and compared with the wild-type structure. Tyr131 is important in the architecture of the active site but is not essential for enzymic activity.
doi:10.1107/S1744309109044741
PMCID: PMC2805523  PMID: 20057057
paAzoR1; azoreductases
6.  Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana  
Overexpression, purification, crystallization and preliminary X-ray diffraction of the stromal-cell-derived factor 2-like protein of Arabidopsis thaliana are reported. The crystals belonged to the space group P61 and diffracted to 1.95 Å resolution.
The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 Å resolution using a synchrotron-radiation source and belonged to the hexagonal space group P61, with unit-cell parameters a = b = 96.1, c = 69.3 Å.
doi:10.1107/S1744309109042018
PMCID: PMC2805525  PMID: 20057059
SDF2-like proteins; unfolded protein response; MIR motifs; β-trefoil domains
7.  Crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 
The MIF4G domain of DAP5 was crystallized in two distinct crystal forms. Diffraction patterns have been analyzed and preliminary analysis, including molecular replacement, is presented here.
Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented.
doi:10.1107/S1744309109044315
PMCID: PMC2805526  PMID: 20057060
DAP5; eIF4G; internal ribosomal entry site; cap-independent translation initiation; translational regulation; eIF4A; eIF3
8.  Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Staphylococcus aureus  
Malonyl-CoA:acyl-carrier protein transacylase (MCAT; FabD) from S. aureus has been cloned, overexpressed, purified and crystallized. The crystal belonged to space group P21, with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 Å, α = γ = 90, β = 106.330°, and data were collected to 1.2 Å resolution using synchrotron radiation.
Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 Å, α = γ = 90, β = 106.330°. The asymmetric unit contains one SaMCAT molecule.
doi:10.1107/S1744309109045989
PMCID: PMC2805527  PMID: 20057061
fatty-acid synthesis; malonyl-CoA:acyl-carrier protein transacylase; FabD; antibacterial drug targets; Staphylococcus aureus
9.  Crystallization and preliminary X-ray analysis of NADH:rubredoxin oxidoreductase from Clostridium acetobutylicum  
NADH:rubredoxin oxidoreductase from C. acetobutylicum was expressed in E. coli, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å.
NADH:rubredoxin oxidoreductase (NROR), an O2-inducible protein, is a versatile electron donor for scavengers of O2 and reactive oxygen species (ROS) in Clostridium acetobutylicum. Recombinant NROR was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the sitting-drop vapour-diffusion method at 293 K. Preliminary crystallo­graphic analysis revealed that the crystals belonged to space group P4122 or P4322, with unit-cell parameters a = b = 98.6, c = 88.3 Å, and diffracted to 2.1 Å resolution. Assuming that the crystals contained one molecule per asymmetric unit, the Matthews coefficient was calculated to be 2.7 Å3 Da−1 and the solvent content to be 54.1%.
doi:10.1107/S1744309109047162
PMCID: PMC2805528  PMID: 20057062
NADH; rubredoxin; oxidoreductases; oxygen response
10.  Crystallization and X-ray diffraction analysis of human CLEC5A (MDL-1), a dengue virus receptor 
Recombinant human CLEC5A was crystallized in the trigonal space group P31 and X-ray diffraction data were collected to 1.56 Å resolution.
The human C-type lectin-like protein CLEC5A (also known as MDL-1) is expressed on the surface of myeloid cells and plays a critical role in dengue-virus-induced disease by signalling through the transmembrane adaptor protein DAP12. The C-type lectin-like domain of CLEC5A was expressed in Escherichia coli, refolded and purified. Recombinant CLEC5A crystals were grown by sitting-drop vapour diffusion using polyethylene glycol 6000 as a precipitant. After optimization, crystals were grown which diffracted to 1.56 Å using synchrotron radiation. The results presented in this paper suggest that crystals producing diffraction of this quality will be suitable for structural determination of human CLEC5A.
doi:10.1107/S1744309109047915
PMCID: PMC2805530  PMID: 20057064
CLEC5A; MDL-1; C-type lectins; DAP12; dengue
11.  Crystallization of dihydrodipicolinate synthase from a clinical isolate of Streptococcus pneumoniae  
The emergence of drug-resistant bacteria highlights the importance of identifying potential drug targets. Dihydrodipicolinate synthase (DHDPS) is a valid but as yet unexploited antimicrobial target that functions in the biosynthesis of (S)-lysine. In this study, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from S. pneumoniae are described.
Dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) catalyzes the rate-limiting step in the (S)-lysine biosynthesis pathway of bacteria and plants. Here, the cloning of the DHDPS gene from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify and crystallize the recombinant enzyme are described. Diffracting crystals were grown in high-molecular-weight PEG precipitants using the hanging-drop vapour-diffusion method. The best crystal, from which data were collected, diffracted to beyond 2.0 Å resolution. Initially, the crystals were thought to belong to space group P42212, with unit-cell parameters a = 105.5, b = 105.5, c = 62.4 Å. However, the R factors remained high following initial processing of the data. It was subsequently shown that the data set was twinned and it was thus reprocessed in space group P2, resulting in a significant reduction in the R factors. Determination of the structure will provide insight into the design of novel antimicrobial agents targeting this important enzyme from S. pneumoniae.
doi:10.1107/S174430910904771X
PMCID: PMC2805531  PMID: 20057065
antimicrobials; antibiotic resistance; dihydrodipicolinate synthase; lysine biosynthesis; Streptococcus pneumoniae
12.  Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase from Escherichia coli  
Diaminopimelate (DAP) epimerase, an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary diffraction analysis of DAP epimerase from E. coli are reported.
Diaminopimelate (DAP) epimerase (EC 5.1.1.7) catalyzes the penultimate step of lysine biosynthesis in bacteria and plants, converting l,l-diaminopimelate to meso-diaminopimelate. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DAP epimerase from Escherichia coli are presented. Crystals were obtained in space group P41212 and diffracted to 2.0 Å resolution, with unit-cell parameters a = b = 89.4, c = 179.6 Å. Molecular replacement was conducted using Bacillus anthracis DAP epimerase as a search model and showed the presence of two molecules in the asymmetric unit, with an initial R free of 0.456 and R work of 0.416.
doi:10.1107/S1744309109047708
PMCID: PMC2805532  PMID: 20057066
antibiotics; antibiotic resistance; cell-wall synthesis; diaminopimelate epimerase; drug discovery; Escherichia coli; lysine biosynthesis
13.  Crystallization and preliminary X-ray crystallographic analysis of the N domain of p97/VCP in complex with the UBX domain of FAF1 
The N domain of p97/VCP was crystallized in complex with the UBX domain of FAF1. X-ray diffraction data were collected to 2.60 Å resolution and the crystals belonged to space group C2221.
p97/VCP is a multifunctional AAA+-family ATPase that is involved in diverse cellular processes. p97/VCP directly interacts with various adaptors for activity in different biochemical contexts. Among these adaptors are p47 and Fas-associated factor 1 (FAF1), which contain a common UBX domain through which they bind to the N domain of p97/VCP. In the ubiquitin–proteasome pathway, p97/VCP acts as a chaperone that presents client proteins to the proteasome for degradation, while FAF1 modulates the process by interacting with ubiquitinated client proteins and also with p97/VCP. In an effort to elucidate the structural details of the interaction between p97/VCP and FAF1, the p97/VCP N domain was crystallized in complex with the FAF1 UBX domain. X-ray data were collected to 2.60 Å resolution and the crystals belonged to space group C2221, with unit-cell parameters a = 58.24, b = 72.81, c = 132.93 Å. The Matthews coefficient and solvent content were estimated to be 2.39 Å3 Da−1 and 48.4%, respectively, assuming that the asymmetric unit contained p97/VCP N domain and FAF1 molecules in a 1:1 ratio, which was subsequently confirmed by molecular-replacement calculations.
doi:10.1107/S1744309109047691
PMCID: PMC2805533  PMID: 20057067
p97; VCP; FAF1; UBX
14.  Crystallization and preliminary X-ray diffraction data of α-galactosidase from Saccharomyces cerevisiae  
α-Galactosidase from S. cerevisiae has been purified and crystallized in glycosylated and deglycosylated states. X-ray diffraction data were collected to 1.95 Å resolution from the deglycosylated form.
Saccharomyces cerevisiae α-galactosidase is a highly glycosylated extracellular protein that catalyzes the hydrolysis of α-galactosidic linkages in various glucids. Its enzymatic activity is of interest in many food-related industries and has biotechnological applications. Glycosylated and in vitro deglycosylated protein samples were both assayed for crystallization, but only the latter gave good-quality crystals that were suitable for X-ray crystallography. The crystals belonged to space group P4212, with unit-cell parameters a = b = 101.24, c = 111.52 Å. A complete diffraction data set was collected to 1.95 Å resolution using a synchrotron source.
doi:10.1107/S1744309109047794
PMCID: PMC2805534  PMID: 20057068
yeast; α-galactosidases; melibiase; glycoside hydrolase family 27; glycosylation
15.  Crystallization and preliminary X-ray crystallographic analysis of DNA gyrase GyrB subunit from Xanthomonas oryzae pv. oryzae  
The GyrB subunit of DNA gyrase from X. oryzae pv. oryzae was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.10 Å.
DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal fragment of the GyrB subunit of DNA gyrase from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.10 Å resolution using a synchrotron-radiation source. The crystal belonged to space group I41, with unit-cell parameters a = b = 110.27, c = 70.75 Å. The asymmetric unit contained one molecule, with a V M of 2.57 Å3 Da−1 and a solvent content of 50.2%.
doi:10.1107/S1744309109047721
PMCID: PMC2805535  PMID: 20057069
DNA gyrase; GyrB subunit; type II topoisomerases; Xanthomonas oryzae pv. oryzae
16.  Crystallization and preliminary X-ray diffraction data analysis of stenodactylin, a highly toxic type 2 ribosome-inactivating protein from Adenia stenodactyla  
Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla. Stenodactylin crystallization and preliminary X-ray diffraction data analysis are reported.
Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15 Å). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit.
doi:10.1107/S1744309109047654
PMCID: PMC2805536  PMID: 20057070
ribosome-inactivating proteins; stenodactylin; Adenia stenodactyla; polynucleotide adenine glycosylases; cytotoxicity
17.  Crystallization and preliminary X-ray crystallographic analysis of Lon from Thermococcus onnurineus NA1 
Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from T. onnurineus NA1 has been purified and crystallized at 295 K.
Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from Thermococcus onnurineus NA1 was purified and crystallized at 295 K. A 2.0 Å resolution data set was collected using synchrotron radiation. The crystals belonged to space group P63, with unit-cell parameters a = 121.45, b = 121.45, c = 195.24 Å. Assuming the presence of two monomers in the asymmetric unit, the solvent content was estimated to be about 60.7%.
doi:10.1107/S1744309109048039
PMCID: PMC2805537  PMID: 20057071
Lon; ATP-dependent proteases; Thermococcus onnurineus NA1
18.  Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus  
Given the recent rise in antimicrobial resistance, there is an urgent need to identify and characterize new antibiotic drug targets. One such target is dihydrodipicolinate reductase (DHDPR), which is an essential bacterial enzyme that catalyzes the second step in the lysine-biosynthesis pathway. In this paper, the cloning, expression, purification and crystallization of DHDPR from methicillin-resistant S. aureus are presented.
Dihydrodipicolinate reductase (DHDPR; EC 1.3.1.26) catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65 Å resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target.
doi:10.1107/S1744309109047964
PMCID: PMC2805538  PMID: 20057072
antibiotic resistance; antimicrobials; dihydrodipicolinate reductase; lysine biosynthesis; MRSA; Staphylococcus aureus
19.  Crystallization and preliminary X-ray crystallographic analysis of Thermus thermophilus transcription elongation complex bound to Gfh1 
To elucidate which RNA polymerase structural state a particular T. thermophilus Gre-family protein (Gfh1) associates with, the T. thermophilus RNAP elongation complex was cocrystallized with Gfh1.
RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P21 with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8 Å resolution, respectively.
doi:10.1107/S1744309109049215
PMCID: PMC2805540  PMID: 20057074
GreA; nucleotide-addition cycle; transcript cleavage
20.  Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461 
Crystals of S. elodea ATCC 31461 UDP-glucose dehydrogenase (EC 1.1.1.22) were obtained in space groups P622 and P43212 and diffracted to 2.4 and 3.4 Å resolution, respectively.
Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 Å resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.
doi:10.1107/S174430910904929X
PMCID: PMC2805541  PMID: 20057075
Sphingomonas elodea; gellan gum; exopolysaccharides; UDP-glucose dehydrogenases
21.  Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein 
A C-terminal truncation construct of human PACSIN 1 (1–344) has been purified and crystallized. Diffraction data were collected to 3.0 Å resolution.
PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1–344) was crystallized and diffracted to a resolution of 3.0 Å. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 Å, α = 90.00, β = 113.61, γ = 90.00°. There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%.
doi:10.1107/S1744309109049549
PMCID: PMC2805542  PMID: 20057076
PACSIN 1
22.  Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution 
Of the four old yellow enzyme homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method.
Shewanella oneidensis is an environmentally versatile Gram-negative γ-proteo­bacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the ortho­rhombic space group P212121 and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 Å resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30–1.60 Å resolution, thus providing a rare opportunity to dissect the structure–function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride–Meisenheimer complex.
doi:10.1107/S1744309109050386
PMCID: PMC2805545  PMID: 20057079
SYE4; Shewanella oneidensis; old yellow enzyme homologues
23.  Crystallization and preliminary X-ray characterization of the Skp1–Fbg3 complex 
The crystallization and preliminary X-ray diffraction studies of the Skp1–Fbg3 complex are reported. Crystallization by repeated microseeding using selected crystals as a source of microseeds is described.
F-box proteins are the substrate-recognition components of Skp1–Cullin1–F-­box protein–Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P212121, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9 Å and one molecule per asymmetric unit.
doi:10.1107/S1744309109050581
PMCID: PMC2805547  PMID: 20057081
F-box proteins; Fbg3; Skp1–Fbg3 complex; ubiquitin ligases
24.  Complex assembly, crystallization and preliminary X-ray crystallographic studies of duck MHC class I molecule 
Using a peptide derived from H5N1, a complex of duck MHC class I molecule (DuMHC I) with duck β2-­microglobulin (Duβ2m) was assembled and crystallized. Initial structure analysis indicated that the crystals did not contain the complete DuMHC I complex but instead contained DuMHC I α3-domain and Duβ2m subunits.
In order to understand the biological properties of the immune systems of waterfowl and to establish a system for structural studies of duck class I major histocompatibility complex (DuMHC I), a complex of DuMHC I with duck β2-­microglobulin (Duβ2m) and the peptide AEIEDLIF (AF8) derived from H5N1 NP residues 251–258 was assembled. The complex was crystallized; the crystals belonged to space group C2221, with unit-cell parameters a = 54.7, b = 72.4, c = 102.2 Å, and diffracted to 2.3 Å resolution. Matthews coefficient calculation and initial structure determination by molecular replacement showed that the crystals did not contain the whole DuMHC I complex, but instead contained the DuMHC I α3 domain and a Duβ2m molecule (DuMHC I α3+β2m). Another complex of DuMHC I with the peptide IDWFDGKE derived from a chicken fusion protein also generated the same results. The stable structure of DuMHC I α3+β2m may reflect some unique characteristics of DuMHC I and pave the way for novel MHC structure-related studies in the future.
doi:10.1107/S174430910905088X
PMCID: PMC2805548  PMID: 20057082
major histocompatibility complex class I molecule; β2-­microglobulin
25.  Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana  
Inositol 1,3,4,5,6-pentakisphosphate kinase from A. thaliana has been expressed in E. coli, purified and crystallized and diffraction data have been collected to 2.3 Å resolution. Two heavy-atom crystal derivatives are under study.
Inositol 1,3,4,5,6-pentakisphosphate kinase (IP5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP5) and ATP. IP6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP6. The best crystals obtained for X-ray crystallographic analysis belonged to space group P212121, with unit-cell parameters a = 58.124, b = 113.591, c = 142.478 Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source.
doi:10.1107/S1744309109051057
PMCID: PMC2805549  PMID: 20057083
inositol kinases; phytic acid; inositol 1,2,3,4,5,6-hexakisphosphate; inositol 1,3,4,5,6-pentakisphosphate kinase

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