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Selected AbstractsThe efficacy of dietetic intervention in patients with chronic obstructive pulmonary diseaseJOURNAL OF HUMAN NUTRITION & DIETETICS, Issue 4 2008L. Bottle Background:, Clinical trials have shown that pulmonary rehabilitation can improve the functional status and quality of life of chronic obstructive pulmonary disease (COPD) patients (Lacasse, 2006) but there is no research examining the efficacy of group dietetic intervention during standard 8 week rehabilitation courses. Current input is usually limited to a 1 h nutrition education session. This pilot study aimed to investigate whether patients receiving additional dietetic intervention during pulmonary rehabilitation significantly increased their general nutritional knowledge, thereby facilitating improvements in dietary intake and nutritional status. Methods:, Patients were recruited from two courses of pulmonary rehabilitation and randomly allocated to a control group or an intervention group. Anthropometry (height, weight, body mass index, mid arm circumference and triceps skinfold), 3 day food diaries and nutritional knowledge questionnaires covered guidelines, food groups, choosing healthy options and diet and COPD were completed at baseline and at the end of 8 weeks. In week 2 both groups received the same nutrition education session which covered healthy eating during periods of stability as well as advice on coping with loss of appetite and reduced intake during illness and exacerbations. The intervention group was followed up during weeks 4, 6 and 7 when further anthropometric measurements were taken and additional dietary advice was provided, which addressed issues raised by individual patients. Information from food diaries was converted to nutrients using Windiets dietary analysis software. Statistical analyses were carried out using SPSS (v14) and included Mann,Whitney U non parametric tests, paired t -tests and Spearman correlations used for comparisons over time and between groups. For analysis purposes patients were classified as normal weight (NW) and overweight (OW). Approval was obtained from the appropriate Ethics Committee. Results:, Changes reported were not statistically significant (P > 0.05). Complete data sets were obtained for six control (NW = 2, OW = 4) and five intervention (NW = 1, OW = 4) patients. Nutritional knowledge increased in the control group by 5% compared to 3% in the intervention group. Control NW patients increased their energy intake resulting in a mean weight gain of 0.5 kg (SD 3.3). OW control group patients increased their energy intake by 12.4% (16.9) with a mean weight gain of 0.2 kg (2.5). All control patients increased their intake of in total fat, saturated fatty acids (SFA), sugars and sodium. Conversely there was a decrease in energy intake in the intervention group of 14.4% (17.8) and a mean weight loss of 1.5 kg (1.2) (three out of four overweight patients lost weight). Improvements in diet were shown with reduced intakes of total fat, SFA, sugars and sodium. The NW patient in the intervention group regained weight that had previously been lost. These changes did not correlate with changes in nutritional knowledge. Discussion:, An increase in nutritional knowledge was expected to facilitate appropriate changes in dietary intake and nutritional status. Despite the lack of correlation between dietary knowledge and intake, beneficial outcomes were none-the-less observed in the intervention group. The trend for weight gain in OW control group patients, and weight loss in OW intervention group patients contrasted with results seen by Slinde et al. (2002) where the control OW patients lost weight, and OW intervention patients gained weight. It is possible that in the current study, patients in the intervention group were motivated to lose weight with repeated exposure to the dietitian, rather than an increase in nutritional knowledge. Significant anthropometrical changes were unlikely to be observed in 8 weeks, and further follow up may be necessary to establish sufficient evidence for the most efficacious level of dietetic intervention. The small sample sizes, especially with regard to weight sub groups, limits the conclusions which can be drawn. Further research is recommended, using a larger sample size, in order to make recommendations for dietetic best practice. Conclusion:, The results of this study did not show statistical significance and the association between nutritional knowledge and improved nutritional outcomes remains unclear. However, the findings may have clinical significance since they appear to show that additional dietetic intervention may benefit the nutritional status of patients with COPD attending pulmonary rehabilitation. References, Lacasse, Y., Goldstein, R., et al. (2006) Pulmonary rehabilitation for chronic obstructive pulmonary disease. Cochrane Database Syst. Rev. 4, CD003793. Slinde, F., Gronberg, A.M., et al. (2002) Individual dietary intervention in patients with COPD during multidisciplinary rehabilitation. Respir. Med. 96, 330,336. [source] Use of a Urine Dipstick and Brief Clinical Questionnaire to Predict an Abnormal Serum Creatinine in the Emergency DepartmentACADEMIC EMERGENCY MEDICINE, Issue 8 2009Daniel N. Firestone MD Abstract Objectives:, Prior data demonstrated that a urine dipstick used alone was a sensitive predictor of abnormal creatinine, but not sufficiently enough to forego screening of serum creatinine prior to administration of contrast for diagnostic studies. The authors hypothesized that a brief historical questionnaire coupled with a urine dipstick would have high sensitivity for renal dysfunction, potentially eliminating the need for a serum creatinine prior to contrast administration. Methods:, This was a prospective study of a convenience sample of patients at two academic tertiary-care emergency departments (EDs) during 2006,2007. Subjects included patients who had both a serum creatinine result reported by the laboratory and a urine dipstick result reported in the medical record. Data included triage vital signs, basic demographic data, 14 medical history items, dipstick urinalysis, and serum creatinine results. The main outcome measure was an abnormal serum creatinine, defined as greater than 1.5 mg/dL. Results:, Complete data sets were collected on 1,354 patient visits. Of these, there were 161 (12%) with a serum creatinine of >1.5 mg/dL. Logistic regression analysis identified the following independent predictors associated with elevated creatinine: age greater than 60 years, known renal insufficiency, diabetes, hypertension, diuretic use, vomiting, and proteinuria. Nearly all patients with abnormal creatinine (98%) had at least one of these seven predictors. A decision tool combining these predictors would have identified 158 of 161 patients with an abnormal creatinine (sensitivity, 98.1%; 95% confidence interval [CI] = 95.8% to 99.9%) and a specificity of 21.2% (95% CI = 18.8% to 23.2%). Conclusions:, The absence of six historical factors and absence of proteinuria can be safely used to identify patients who are unlikely to have an abnormal creatinine. [source] Overproduction, crystallization and preliminary crystallographic analysis of a novel human DNA-repair enzyme that recognizes oxidative DNA damageACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004Viswanath Bandaru DNA glycosylases repair oxidative DNA damage caused by free radicals. Recently, NEIL1, a human homolog of Escherichia coli DNA glycosylase endonuclease VIII, has been identified and shown to exhibit broad substrate specificity for a variety of types of pyrimidine-base damage. An active C-terminal deletion construct of NEIL1 was overexpressed in E. coli and crystallized. The unliganded NEIL1 crystallizes in space group R3, with unit-cell parameters a = b = 132.2, c = 51.1,Å. Complete data sets were collected from native, selenomethionyl and iodinated NEIL1 to 2.1, 2.3 and 2.4,Å, respectively. [source] Expression, purification, crystallization and preliminary crystallographic analysis of phosphoserine aminotransferase from Bacillus alcalophilusACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Anatoly P. Dubnovitsky Phosphoserine aminotransferase (PSAT; EC 2.6.1.52) from Bacillus alcalophilus, an obligatory alkalophile with optimum growth at pH 10.6, was overexpressed in Escherichia coli, purified and crystallized under two different conditions using the hanging-drop vapour-diffusion method. Crystals were obtained using trisodium citrate dihydrate or PEG 400 as a precipitating agent. Crystals grown in the presence of trisodium citrate belong to the orthorhombic space group C2221, with unit-cell parameters a = 105.6, b = 136.6, c = 152.0,Å, and those grown in the presence of PEG 400 belong to the orthorhombic space group P21212, with unit-cell parameters a = 143.7, b = 84.3, c = 67.4,Å. Complete data sets were collected to 1.7 and 1.6,Å resolution, respectively, at 100,K using synchrotron radiation. Analysis of the structure of B. alcalophilus PSAT may reveal structural features that contribute to enzyme adaptability at high pH values. [source] Overproduction, crystallization and preliminary diffraction data of ADP-ribose pyrophosphatase from Thermus thermophilus HB8ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003Sachiko Yoshiba An ADP-ribose pyrophosphatase from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography showed the protein to be in a dimeric state. This protein catalyses the Mg2+ - or Zn2+ -dependent hydrolysis of ADP-ribose to AMP and ribose-5,-phosphate. It was crystallized in the absence and the presence of ADP-ribose by the hanging-drop vapour-diffusion method. Complete data sets were collected to 1.50,Å resolution from the apo form using synchrotron radiation and to 2.0,Å resolution from the complexed form. Both crystals belong to space group P3121 or P3221 and contain one molecule in the asymmetric unit. [source] Cloning, expression, purification and crystallization of the N-domain from the ,2 subunit of the membrane-spanning Na,K-ATPase proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Lisbeth Haue The nucleotide-binding domain of the Na,K-ATPase ion pump was expressed with a His tag in Escherichia coli and purified. The soluble 24,kDa derivative consists of 214 amino-acid residues and was crystallized in the presence of NiCl2. The crystals belong to space group F23, with unit-cell parameters a = b = c = 147.5,Å, and diffract to 3.1,Å. Complete data sets could be collected from native and thimerosal-treated crystals frozen in 50% sucrose. Five mercury positions were found and initial SIR phases calculated. [source] Crystallization of the Mycobacterium tuberculosis cell-division protein FtsZACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000Adelaine K. W. Leung Mycobacterium tuberculosis FtsZ (MtbFtsZ), an essential protein in bacterial cell division, has been crystallized in the presence of a new inhibitor of MtbFtsZ polymerization and GTPase activity, ethyl (6-amino-2,3-dihydro-4-phenyl-1H -pyrido[4,3- b][1,4]diazepin-8-yl)carbamate (SRI-7614). Crystals of the MtbFtsZ,SRI-7614 complex (form I, 30% polyethylene glycol 4000, 0.1,M sodium citrate pH 5.6, 0.2,M NH4OAc, 293,K) belong to space group P61 or P65, with unit-cell parameters a = 88.78, c = 178.02,Å, and diffract to 2.3,Å resolution. A second crystal form, of the GDP complex, grows in the presence or absence of Mg2+ from PEG 4000 at 277,K or from (NH4)2SO4 at 293,K, respectively (form II, space group P6222 or P6422, with unit-cell parameters a = 135.02, c = 328.97,Å or a = 129.30, c = 327.97,Å, respectively). Complete data sets to ,7,Å resolution have been collected from both. Exceptional form II crystals diffract to at least 4.5,Å resolution. Determination of the MtbFtsZ structure may advance the design of improved inhibitors of FtsZ polymerization. [source] Crystallization and preliminary X-ray analysis of a d -alanyl- d -alanine ligase (EcDdlB) from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Sarah Batson A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and d -alanyl- d -alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and d -alanyl- d -alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5,Å and a = 51.2, b = 97.8, c = 110.1,Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4,Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation. [source] Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ,29ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2005Dwight L. Anderson The Bacillus subtilis bacteriophage ,29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293,K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12,Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34,Å. Complete data sets have been collected to 1.78 and 1.80,Å for forms I and II, respectively, at 100,K using Cu,K, X-rays from a rotating-anode generator. Calculation of a VM value of 2.46,Å3,Da,1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80,Å3,Da,1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7. [source] Alternative approaches can greatly reduce the number of fish used for acute toxicity testingENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2006Conny C. Hoekzema Abstract Acute toxicity tests with algae, daphnids, and fish are required for the classification and environmental risk assessment of chemicals. The degree of risk is determined by the lowest of these acute toxicity values. Many ecotoxicological programs are seeking to reduce the numbers of fish used in acute toxicity testing. The acute threshold test is a recently proposed strategy that uses, on average, only 10 (instead of 54) fish per chemical. We examined the consequences of reducing the number of fish used in toxicity testing on the ultimate outcome of risk assessments. We evaluated toxicity data sets for 507 compounds, including agrochemicals, industrial chemicals, and pharmaceuticals from our internal database. Theoretical applications of the acute threshold test gave similar results to those obtained with the standard fish median lethal concentration (LC50) test but required only 12% as many fish (3,195 instead of 27,324 fish used for all compounds in the database). In 188 (90%) of the 208 cases for which a complete data set was available, the median effect concentration for algae or daphnids was lower than the LC50 for fish. These results show that replacement of the standard fish LC50 test by the acute threshold test would greatly reduce the number of fish needed for acute ecotoxicity testing without any loss of reliability. [source] Forming professional identities on the health care team: discursive constructions of the ,other' in the operating roomMEDICAL EDUCATION, Issue 8 2002L Lingard Background, Inter-professional health care teams represent the nucleus of both patient care and the clinical education of novices. Both activities depend upon the,talk' that team members use to interact with one another. This study explored team members' interpretations of tense team communications in the operating room (OR). Methods, The study was conducted using 52 team members divided into 14 focus groups. Team members comprised 13 surgeons, 19 nurses, nine anaesthetists and 11 trainees. Both uni-disciplinary (n = 11) and multi-disciplinary (n = 3) formats were employed. All groups discussed three communication scenarios, derived from prior ethnographic research. Discussions were audio-recorded and transcribed. Using a grounded theory approach, three researchers individually analysed sample transcripts, after which group discussions were held to resolve discrepancies and confirm a coding structure. Using the confirmed code, the complete data set was coded using the ,NVivo' qualitative data analysis software program. Results, There were substantial differences in surgeons', nurses', anaesthetists', and trainees' interpretations of the communication scenarios. Interpretations were accompanied by subjects' depictions of disciplinary roles on the team. Subjects' constructions of other professions' roles, values and motivations were often dissonant with those professions' constructions of themselves. Conclusions, Team members, particularly novices, tend to simplify and distort others' roles and motivations as they interpret tense communication. We suggest that such simplifications may be rhetorical, reflecting professional rivalries on the OR team. In addition, we theorise that novices' echoing of role simplification has implications for their professional identity formation. [source] ST-Segment Resolution Prior to Primary Percutaneous Coronary Intervention Is a Poor Indicator of Coronary Artery Patency in Patients with Acute Myocardial InfarctionANNALS OF NONINVASIVE ELECTROCARDIOLOGY, Issue 2 2010Niels J. Verouden M.D. Background: The prognostic value of ST-segment resolution (STR) after initiation of reperfusion therapy has been established by various studies conducted in both the thrombolytic and mechanic reperfusion era. However, data regarding the value of STR immediately prior to primary percutaneous coronary intervention (PCI) to predict infarct-related artery (IRA) patency remain limited. We investigated whether STR prior to primary PCI is a reliable, noninvasive indicator of IRA patency in patients with ST-segment elevation myocardial infarction (STEMI). Methods: The study population consisted of STEMI patients who underwent primary PCI at our institution between 2000 and 2007. STR was analyzed in 12-lead electrocardiograms recorded at first medical contact and immediately prior to primary PCI and defined as complete (,70%), partial (70%, 30%), or absent (<30%). Results: In 1253 patients with a complete data set, STR was inversely related to the probability of impaired preprocedural flow (Pfor trend < 0.001). Although the sensitivity of incomplete (<70%) STR to predict a Thrombolysis in Myocardial Infarction (TIMI) flow of <3 was 96%, the specificity was 23%, and the negative predictive value of incomplete STR to predict normal coronary flow was only 44%. Conclusions: This study establishes the correlation between STR prior to primary PCI and preprocedural TIMI flow in STEMI patients treated with primary PCI. However, the negative predictive value of incomplete STR for detection of TIMI-3 flow is only 44% and therefore should not be a criterion to refrain from immediate coronary angiography in STEMI patients. Ann Noninvasive Electrocardiol 2010;15(2):107,115 [source] The minimum crystal size needed for a complete diffraction data setACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010James M. Holton In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2,Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2,µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34,µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits. [source] Feasibility of one-shot-per-crystal structure determination using Laue diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2010Sterling Cornaby Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a `pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20,30,µm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used. [source] A systematic study of 50S ribosomal subunit purification enabling robust crystallizationACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Thomas J. McLellan A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9,Å resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested. [source] Crystallization and preliminary X-ray crystallographic analysis of the laminarinase endo-,-1,3-glucanase from Pyrococcus furiosusACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Andrea Ilari Laminarinase endo-,-1,3 glucanase (LamA) from Pyrococcus furiosus is an enzyme which displays its main hydrolytic activity on the ,-1,3-glucose polymer laminarin. This laminarinase is remarkably resistant to denaturation: its secondary structure is unchanged in 8,M guanidinium chloride. This protein belongs to the family 16 glycosyl hydrolases, which are enzymes that are widely distributed among bacteria, fungi and higher plants. Single crystals of P. furiosus LamA have been obtained by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol as a precipitant agent. A complete data set has been collected under cryocooling at a synchrotron source. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 44.36, b = 84.76, c = 69.23,Å, , = 90, , = 104.97, , = 90°, and diffract to 2.15,Å resolution. [source] Purification, crystallization, X-ray diffraction analysis and phasing of a Fab fragment of monoclonal neuroantibody ,D11 against nerve growth factorACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Sonia Covaceuszach The rat monoclonal neuroantibody ,D11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of ,D11 were performed. ,D11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The ,D11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7,Å, , = 82.0, , = 89.1, , = 86.0°. With two molecules in the asymmetric unit, VM is 2.3,Å3,Da,1 and the solvent content is 46%. A complete data set has been collected at 2.7,Å resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10,Å, , = 117.0°. With one molecule in the asymmetric unit, VM is 2.4,Å3,Da,1 and the solvent content is 48%. A complete data set has been collected at 1.7,Å resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern. [source] Purification, crystallization and preliminary X-ray diffraction analysis of human oncoprotein SET/TAF-1,ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Shinsuke Muto The human oncoprotein SET/TAF-1, has been crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 119.6, b = 62.8, c = 61.0,Å, , = 89.7°, and contains two molecules in the asymmetric unit. A complete data set was collected to 2.8,Å resolution using synchrotron radiation. [source] Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Jieqing Zhu Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers [(HR1,HR2)3] after the fusion between viral and cellular membranes. In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli. Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method. A complete data set was collected in-house to 1.9,Å resolution from a single crystal. The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058,Å. To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8,Å using synchrotron radiation. [source] Definition of domain boundaries and crystallization of the SMN Tudor domainACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003Remco Sprangers Spinal muscular atropy (SMA) is the major genetic disease leading to childhood mortality and is caused by mutations in or deletions of the smn1 gene. The human survival of motor neurons (SMN) protein encoded by this gene plays an important role in the assembly of snRNPs (small nuclear ribonucleoprotein complexes) via binding to the spliceosomal Sm proteins. The tails of these Sm proteins contain symmetrically dimethylated arginines that are recognized by the central SMN Tudor domain. To gain insight in the molecular basis of this specific interaction, the SMN Tudor domain has been crystallized. The rapid crystallization of the protein and the high stability of the crystals is facilitated by redefinition of domain boundaries based on NMR relaxation experiments and the previously determined solution structure. The crystals diffract to high resolution (1.8,Å) and a complete data set has been collected from a hexagonal crystal form (P61/P65), with unit-cell parameters a = b = 27.65, c = 110.30,Å, , = , = 90, , = 120°. Crystal soaks and co-crystallization with peptides derived from the Sm protein tails have been initiated. Molecular replacement with the NMR coordinates is under way. [source] Crystallization and preliminary crystallographic analysis of a family 45 endoglucanase from the thermophilic fungus Melanocarpus albomycesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Mika Hirvonen 20K-cellulase, an endoglucanase produced by the thermophilic fungus Melanocarpus albomyces, has been crystallized. It belongs to the family 45 of glycoside hydrolases and has sequence homology with Humicola insolens endoglucanase V (EGV). However, in contrast to EGV, it does not harbour a cellulose-binding module. Optimization of the crystallization conditions using a PEG/ion combination and microseeding techniques was employed to improve the quality and the size of the crystals. A complete data set to 2.2,Å resolution was collected using synchrotron radiation. Preliminary crystallographic analysis showed that the crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = 47.3, b = 47.3, c = 177.3,Å and one molecule per asymmetric unit. [source] Crystallization of the Z, domain of the human editing enzyme ADAR1 complexed with a DNA,RNA chimeric oligonucleotide in the left-handed Z-conformationACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Bernard A. Brown II The Z, domain of human double-stranded RNA adenosine deaminase (ADAR1) has been crystallized with a hexanucleotide containing alternating deoxyribose and ribose furanose sugars. Solution circular dichroism experiments show that this double-stranded chimera (dCrG)3 initially adopts the right-handed A-conformation. However, addition of stoichiometric amounts of Z, causes a rapid transition to the Z-conformation. Raman spectroscopy of crystals of the Z,,(dCrG)3 complex confirm that the chimeric oligonucleotide is stabilized in the Z-conformation. A complete data set has been obtained at 2.5,Å resolution. The Z,,(dCrG)3 crystals belong to the tetragonal I422 space group, with unit-cell parameters a = b = 104.2, c = 117.6,Å. Work is under way to solve the structure by molecular replacement. [source] Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132GlyACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Hiroaki Nakano Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84,85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2,Å resolution using synchrotron radiation at 100,K. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 46.02, b = 49.27, c = 49.37,Å, , = 110.1°. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation. [source] Crystallization and preliminary X-ray crystallographic studies of the thermoactive pullulanase type I, hydrolyzing ,-1,6 glycosidic linkages, from Fervidobacterium pennivorans Ven5ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Joyce H. G. Lebbink Crystals of the thermoactive recombinant F. pennivorans type I pullulanase, purified from the supernatant of a Bacillus subtilis culture, have been obtained by the vapour-diffusion method in the presence of the inhibitor ,-cyclodextrin (2,mM) by mixing protein (15,mg,ml,1) with an equal volume of crystallization solution containing 0.1,M bis,tris propane pH 6.5, 50,mM MgCl2 and 15% polyethylene glycol 3350. Crystals diffracted to 3.0,Å using conventional Cu,K, radiation and belong to space group P212121, with unit-cell parameters a = 76.8, b = 96.2, c = 98.5,Å. The asymmetric unit contains one monomer. A preliminary 26% complete data set has been collected at 2.2,Å resolution using synchrotron radiation. [source] Improved crystals of Thermus thermophilus prolyl-tRNA synthetase complexed with cognate tRNA obtained by crystallization from precipitateACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2000Anna Yaremchuk The complex between Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) and its cognate tRNA has been crystallized using two different isoacceptors of tRNAPro. Similar bipyramidal crystals of the complexes of ProRSTT with the two different tRNAPro isoacceptors grow within two weeks from 32% saturated ammonium sulfate solution. They belong to space group P43212, with unit-cell parameters a = 143.1, b = 143.1, c = 228.6,Å. The crystals diffract weakly to a maximum resolution of 3.1,Å. Superior quality crystals were obtained by growing slowly from precipitate over 5,6 months. These are of the same space group but have slightly altered unit-cell parameters, a = 140.8, b = 140.8, c = 237.0,Å. These crystals diffract more strongly to at least 2.8,Å resolution and a complete data set to 2.85,Å resolution has been collected from a single crystal. Comparison of the packing in the two crystal forms shows that domain flexibility contributes to the presence of different crystal contacts in the two forms. [source] Preliminary crystallographic analysis of the Escherichia coli antitoxin MqsA (YgiT/b3021) in complex with mqsRA promoter DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Breann L. Brown The Escherichia coli proteins MqsR and MqsA comprise a novel toxin,antitoxin (TA) system. MqsA, the antitoxin, defines a new family of antitoxins because unlike other antitoxins MqsA is structured throughout its entire sequence, binds zinc and coordinates DNA via its C-terminal and not its N-terminal domain. In order to understand how bacterial antitoxins, and MqsA in particular, regulate transcription, the MqsA protein was cocrystallized with a 26-mer duplex DNA corresponding to the palindromic region of the mqsRA promoter. The merohedrally twinned crystal belonged to space group P41, with unit-cell parameters a = 60.99, b = 60.99, c = 148.60,Å. A complete data set was collected to a resolution of 2.1,Å. The solvent content of the crystal was consistent with the presence of two MqsA molecules bound to the duplex DNA in the asymmetric unit. [source] Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Paul T. Harris Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25,Å resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79,Å. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8,Å3,Da,1, corresponding to 55.2% solvent content. [source] Expression, purification, crystallization and preliminary X-ray analysis of the EIICGlc domain of the Escherichia coli glucose transporterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Andreas Zurbriggen The glucose-import system of Escherichia coli consists of a hydrophilic EIIAGlc subunit and a transmembrane EIICBGlc subunit. EIICBGlc (UniProt P69786) contains two domains: the transmembrane EIICGlc domain (40.6,kDa) and the cytoplasmic EIIBGlc domain (8.0,kDa), which are fused by a linker that is strongly conserved among its orthologues. The EIICBGlc subunit can be split within this motif by trypsin. Here, the crystallization of the tryptic EIICGlc domain is described. A complete data set was collected to 4.5,Å resolution at 100,K. [source] Purification, crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltF from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Pramod K. Madoori The lytic transglycosylase MltF from Escherichia coli is an outer-membrane-bound periplasmic protein with two domains: a C-terminal catalytic domain with a lysozyme-like fold and an N-terminal domain of unknown function that is homologous to the periplasmic substrate-binding proteins of ABC transporters. In order to investigate its structure and function, a soluble form of full-length MltF (sMltF) containing both domains and a soluble fragment containing only the N-terminal domain (sMltF-NTD) were purified and crystallized. Crystals of sMltF belonged to space group P43212 or P41212, with unit-cell parameters a = b = 110.8, c = 163.5,Å and one or two molecules per asymmetric unit. A complete data set was collected to 3.5,Å resolution. Crystals of sMltF-NTD belonged to space group P3121, with unit-cell parameters a = b = 82.4, c = 75.2,Å and one molecule per asymmetric unit. For sMltF-NTD, a complete native data set was collected to 2.20,Å resolution. In addition, for phasing purposes, a three-wavelength MAD data set was collected to 2.5,Å resolution using a bromide-soaked sMltF-NTD crystal. Using phases derived from the Br-MAD data, it was possible to build a partial model of sMltF-NTD. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-terminal carbohydrate-recognition domain of human galectin-4ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Ana Lucia L. R. Zimbardi Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2,Å resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6122, with unit-cell parameters a = b = 71.25, c = 108.66,Å. The asymmetric unit contained one molecule of hGal4-CRD-1, with a VM value of 2.34,Å3,Da,1 and a solvent content of 47.51%. [source] | |||||||||||||||||||