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Diffraction-quality Crystals (diffraction-quality + crystal)
Selected AbstractsThe effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2007James A. Letts The human ABO(H) blood-group antigens are oligosaccharide structures that are expressed on erythrocyte and other cell surfaces. The terminal carbohydrate residue differs between the blood types and determines the immune reactivity of this antigen. Individuals with blood type A have a terminal N -acetylgalactosamine residue and those with blood type B have a terminal galactose residue. The attachment of these terminal carbohydrates are catalyzed by two different glycosyltransferases: an ,(1,3)N -acetylgalactosaminyltransferase (GTA) and an ,(1,3)galactosyltransferase (GTB) for blood types A and B, respectively. GTA and GTB are homologous enzymes that differ in only four of 354 amino-acid residues (Arg/Gly176, Gly/Ser235, Leu/Met266 and Gly/Ala268 in GTA and GTB, respectively). Diffraction-quality crystals of GTA and GTB have previously been grown from as little as 10,mg,ml,1 stock solutions in the presence of Hg, while diffraction-quality crystals of the native enzymes require much higher concentrations of protein. The structure of a single mutant C209A has been determined in the presence and absence of heavy atoms and reveals that when mercury is complexed with Cys209 it forces a significant level of disorder in a polypeptide loop (amino acids 179,195) that is known to cover the active site of the enzyme. The observation that the more highly disordered structure is more amenable to crystallization is surprising and the derivative provides insight into the mobility of this polypeptide loop compared with homologous enzymes. [source] Crystallization and preliminary X-ray diffraction studies of the water-soluble state of the pore-forming toxin sticholysin II from the sea anemone Stichodactyla helianthusACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002José M. Mancheño Sticholysin II (StnII) is a potent cytolytic protein produced by the sea anemone Stichodactyla helianthus. StnII belongs to the actinoporin family, a group of proteins which are characterized by their ability to spontaneously interact with biological membranes. The cytolytic character of these proteins is currently explained in terms of a molecular mechanism involving the formation of transmembrane pores. StnII has been crystallized using the hanging-drop vapour-diffusion method at 291,K. Diffraction-quality crystals have unit-cell parameters a = 32.30, b = 119.73, c = 43.42,Å, , = 90.04° and belong to the monoclinic space group P21. Diffraction data to a resolution of 1.71,Å were collected at synchrotron facilities. [source] Crystallization and preliminary X-ray analysis of UDP- N -acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureusACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2001Melissa S. Harris UDP- N -acetylenolpyruvylglucosamine reductase (MurB) is an essential enzyme in the bacterial cell-wall biosynthetic pathway, making it a potential therapeutic target for novel antibiotics. Diffraction-quality crystals of both the native and Se-methionine-expressed MurB from Staphylococcus aureus have been prepared by sitting-drop vapour diffusion from solutions containing polyethylene glycol (PEG) 8000, ammonium sulfate, sodium cacodylate pH 6.5 and dimethyl sulfoxide (DMSO). Crystals belong to the cubic space group I213, with unit-cell parameters a = b = c = 178.99,Å. X-ray data from these crystals were collected at the Advanced Photon Source 17-ID beamline and were used to solve the MurB structure to 2.3,Å resolution. [source] Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Wang-Hong Zhao The SMU.2055 gene from the major caries pathogen Streptococcus mutans is annotated as a putative acetyltransferase with 163 amino-acid residues. In order to identify its function via structural studies, the SMU.2055 gene was cloned into the expression vector pET28a. Native and SeMet-labelled SMU.2055 proteins with a His6 tag at the N-terminus were expressed at a high level in Escherichia coli strain BL21 (DE3) and purified to homogeneity by Ni2+ -chelating affinity chromatography. Diffraction-quality crystals of SeMet-labelled SMU.2055 were obtained using the sitting-drop vapour-diffusion method and diffracted to a resolution of 2.5,Å on beamline BL17A at the Photon Factory, Tsukuba, Japan. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 92.0, b = 95.0, c = 192.2,Å. The asymmetric unit contained four molecules, with a solvent content of 57.1%. [source] Crystallization and preliminary X-ray analysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Sandeep Chhabra 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg2+ -dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65,Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 36.8, b = 76.6, c = 51.5,Å, , = , = 90.0, , = 100.2°. The crystals contained two molecules per asymmetric unit, with a volume per protein weight (VM) of 2.04,Å3,Da,1 and an estimated solvent content of 39.6%. [source] Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Tatsuki Ebisawa Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20,Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89,Å, , = 90.96, , = 103.41, , = 101.79°. The Matthews coefficient (VM = 2.10,Å3,Da,1) indicated that the crystal contained two mAG molecules per asymmetric unit. [source] Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogasterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Yumiko Mishima Gram-negative bacteria-binding protein 3 (GNBP3) is a pattern-recognition receptor which contributes to the defensive response against fungal infection in Drosophila. The protein consists of an N-terminal domain, which is considered to recognize ,-glucans from the fungal cell wall, and a C-terminal domain, which is homologous to bacterial glucanases but devoid of activity. The N-terminal domain of GNBP3 (GNBP3-Nter) was successfully purified after expression in Drosophila S2 cells. Diffraction-quality crystals were produced by the hanging-drop vapour-diffusion method using PEG 2000 and PEG 8000 as precipitants. Preliminary X-ray diffraction analysis revealed that the GNBP3-Nter crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 134.79, b = 30.55, c = 51.73,Å, , = 107.4°, and diffracted to 1.7,Å using synchrotron radiation. The asymmetric unit is expected to contain two copies of GNBP3-Nter. Heavy-atom derivative data were collected and a samarium derivative showed one high-occupancy site per molecule. [source] Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Xiaotian Zhong CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67,kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P32, with unit-cell parameters a = b = 118.1, c = 81.6,Å and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2,Å data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P32 lattice and rigid-body refined and position-minimized with PHENIX. [source] Purification, crystallization and preliminary X-ray diffraction analysis of human Gadd45,ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Wenzheng Zhang Gadd45, MyD118 and CR6 (also termed Gadd45,, Gadd45, and Gadd45,, respectively) comprise a family of proteins that play important roles in negative growth control, maintenance of genomic stability, DNA repair, cell-cycle control and apoptosis. Recombinant human Gadd45, and its selenomethionine derivative were expressed in an Escherichia coli expression system and purified; they were then crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 291,K using PEG 3350 as precipitant. Using synchrotron radiation, the best diffraction data were collected to 2.3,Å resolution for native crystals at 100,K; selenomethionyl derivative data were collected to 3.3,Å resolution. All the crystals belonged to space group I213, with approximate unit-cell parameters a = b = c = 126,Å. [source] Cloning, expression, purification, crystallization and preliminary X-ray studies of epoxide hydrolases A and B from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2006Bichitra K. Biswal Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1,Å resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA. [source] Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Kavyashree Manjunath The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05,M cadmium sulfate hydrate, 0.1,M HEPES buffer pH 7.5 and 1.0,M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13,Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (VM) of 2.3,Å3,Da,1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides. [source] Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Akihiro Yamamura Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d -pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7,Å resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P212121, with unit-cell parameters a = 55.02, b = 68.30, c = 68.93,Å. The Matthews coefficient (VM = 1.76,Å3,Da,1) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit. [source] Clear strategy screens for macromolecular crystallizationJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2001Andrzej Marek Brzozowski The development of high-throughput crystallography combined with the wealth of already accumulated information about protein crystallization properties requires constant revision of current crystallization screening procedures. Two complementary 6 × 4 matrix `clear strategy screens' (CSS) have been developed and tested on a number of previously non-crystallized proteins. The screens yielded diffraction-quality crystals of a wide range of proteins (enzymes, transcription factors, structural proteins, etc.) in cases where the applications of commercially available screens were unsuccessful. Both their inherently simple design and their flexible nature provide an experimenter with a logical platform for further modification and optimization. Furthermore, the screens facilitate cryoprotection and potential incorporation of anomalous scatterers for multiple/single-wavelength anomalous dispersion (MAD/SAD) experiments. [source] Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehydeACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Matthew W. Vetting The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane,dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6,Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68,Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups. [source] Improved classification of crystallization images using data fusion and multiple classifiersACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2008Samarasena Buchala Identifying the conditions that will produce diffraction-quality crystals can require very many crystallization experiments. The use of robots has increased the number of experiments performed in most laboratories, while in structural genomics centres tens of thousands of experiments can be produced every day. Reliable automated evaluation of these experiments is becoming increasingly important. A more robust classification is achieved by combining different methods of feature extraction with the use of multiple classifiers. [source] The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2007James A. Letts The human ABO(H) blood-group antigens are oligosaccharide structures that are expressed on erythrocyte and other cell surfaces. The terminal carbohydrate residue differs between the blood types and determines the immune reactivity of this antigen. Individuals with blood type A have a terminal N -acetylgalactosamine residue and those with blood type B have a terminal galactose residue. The attachment of these terminal carbohydrates are catalyzed by two different glycosyltransferases: an ,(1,3)N -acetylgalactosaminyltransferase (GTA) and an ,(1,3)galactosyltransferase (GTB) for blood types A and B, respectively. GTA and GTB are homologous enzymes that differ in only four of 354 amino-acid residues (Arg/Gly176, Gly/Ser235, Leu/Met266 and Gly/Ala268 in GTA and GTB, respectively). Diffraction-quality crystals of GTA and GTB have previously been grown from as little as 10,mg,ml,1 stock solutions in the presence of Hg, while diffraction-quality crystals of the native enzymes require much higher concentrations of protein. The structure of a single mutant C209A has been determined in the presence and absence of heavy atoms and reveals that when mercury is complexed with Cys209 it forces a significant level of disorder in a polypeptide loop (amino acids 179,195) that is known to cover the active site of the enzyme. The observation that the more highly disordered structure is more amenable to crystallization is surprising and the derivative provides insight into the mobility of this polypeptide loop compared with homologous enzymes. [source] Structure of Escherichia coli tryptophanaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006Shao-Yang Ku Pyridoxal 5,-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the ,-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the ,-proton of the substrate for ,-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate,PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal. [source] Co-crystallization of Leptospira interrogans peptide deformylase with a potent inhibitor and molecular-replacement schemes with eight subunits in an asymmetric unitACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Peptide deformylase Translation initiation in eubacteria involves a formylmethionine at the N-terminus of newly synthesized polypeptides. This N-formyl group is removed by peptide deformylase (PDF) during the post-translation process. Such a formylation/deformylation cycle is essential for the cell survival of eubacteria, but is not utilized in eukaryotic cytosolic protein biosynthesis. In view of the absence of deformylase activity in mammalian cells, this is an attractive target for the design of novel antibiotic drugs. Co-crystallization of peptide deformylase from Leptospira interrogans (LiPDF) with its natural inhibitor actinonin produced diffraction-quality crystals that belong to space group P21, with unit-cell parameters a = 87.5, b = 119.1, c = 95.8,Å, , = 111.6°. The 3.1,Å resolution data set collected in-house was used to obtain phases by molecular replacement. Three schemes for the correction of the preliminary solutions were proposed and proved successful in determining the structure of LiPDF with eight subunits in the asymmetric unit. [source] Protein crystallization for genomics: towards high-throughput optimization techniquesACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Naomi E. Chayen Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X-ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X-ray analysis and bioinformatics stages once diffraction-quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large-scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high-throughput techniques. [source] Crystallization and preliminary X-ray study of an N-terminal fragment of rat liver ribosomal P2 proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002David Mandelman Ribosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)2,P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1,65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4,Å resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0,Å. [source] Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Tania F. Oliveira Glutamate dehydrogenase (EC 1.4.1.2,4) from Peptoniphilus asaccharolyticus has been expressed as a selenomethionine-derivatized recombinant protein and diffraction-quality crystals have been grown that are suitable for structure determination. Preliminary structural analyses indicate that the protein assembles as a homohexameric enzyme complex in solution, similar to other bacterial and mammalian enzymes to which its sequence identity varies between 25 and 40%. The structure will provide insight into its preference for the cofactor NADH (over NADPH) by comparisons with the known structures of mammalian and bacterial enzymes. [source] Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgXACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Joel T. Weadge AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9,Å, , = 95.7°. The crystals exhibited the symmetry of space group P21 and diffracted to a minimum d -spacing of 2.1,Å. On the basis of the Matthews coefficient (VM = 2.25,Å3,Da,1), two molecules were estimated to be present in the asymmetric unit. [source] The taming of small heat-shock proteins: crystallization of the ,-crystallin domain from human Hsp27ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009E. V. Baranova Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones. sHsps function as homooligomers or heterooligomers that are prone to subunit exchange and structural plasticity. Here, a procedure for obtaining diffraction-quality crystals of the ,-crystallin domain of human Hsp27 is presented. Initially, limited proteolysis was used to delineate the corresponding stable fragment (residues 90,171). This fragment could be crystallized, but examination of the crystals using X-rays indicated partial disorder. The surface-entropy reduction approach was applied to ameliorate the crystal quality. Consequently, a double mutant E125A/E126A of the 90,171 fragment yielded well ordered crystals that diffracted to 2.0,Å resolution. [source] Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA elementACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Nithya Baburajendran The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7,Å resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2,M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78,Å, , = , = , = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit. [source] Expression, refolding, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgEACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009John C. C. Whitney AlgE is an outer membrane protein present in alginate-producing (mucoid) Pseudomonas aeruginosa. AlgE has been overexpressed in insoluble inclusion bodies, purified under denaturing conditions and refolded in a buffer containing decyl ,- d -maltopyranoside. Purified refolded AlgE was detergent-exchanged into n -octyl tetraoxyethylene and diffraction-quality crystals were grown using the hanging-drop vapor-diffusion method. The crystals grew as small hexagons with unit-cell parameters a = 98.8, b = 156.8, c = 90.4,Å, , = , = , = 90.0°. The crystals exhibited the symmetry of space group C222 and diffracted to a minimum d -spacing of 3.0,Å. On the basis of the Matthews coefficient (VM = 3.28,Å3,Da,1), one molecule is estimated to be present in the asymmetric unit. [source] Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studiesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Thomas F. Lerch Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication. [source] Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas fluorescens AlgKACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2007Carrie-Lynn Keiski AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15,Å, , = 96.97°. The crystals exhibit the symmetry of space group P21 and diffract to a minimum d -spacing of 2.5,Å at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (VM = 2.53,Å3,Da,1), four protein molecules are estimated to be present in the asymmetric unit. [source] | ||||||||||