scholarly journals Crystal structure of the starch-binding domain of glucoamylase fromAspergillus niger

2017 ◽  
Vol 73 (10) ◽  
pp. 550-554 ◽  
Author(s):  
Yousuke Suyama ◽  
Norifumi Muraki ◽  
Masami Kusunoki ◽  
Hideo Miyake
Keyword(s):  
Disulfide Bond ◽  
Expression System ◽  
Binding Domain ◽  
Map Comparison ◽  
X Ray ◽  
Replacement Method ◽  
Glucose Syrup ◽  
Significant Difference ◽  
Molecular Replacement Method ◽  
The Difference

Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase fromAspergillus nigeris a small globular protein containing a disulfide bond. The structure ofA. nigerSBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from anEscherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Å resolution. The SBD consisted of seven β-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each β-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the Cαatom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, theBfactors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis

2015 ◽  
Vol 71 (4) ◽  
pp. 466-470 ◽  
Author(s):  
Ruyi Ding ◽  
Cui Xu ◽  
Xu Chen ◽  
Mengyun Bao ◽  
Xiaoting Qiu
Keyword(s):  
Diffraction Analysis ◽  
Biosynthetic Pathway ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Antithrombotic Activity ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosawith a C-terminal His6tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space groupP4322, with unit-cell parametersa=b= 101.581,c= 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Rgg protein structure–function and inhibition by cyclic peptide compounds

2015 ◽  
Vol 112 (16) ◽  
pp. 5177-5182 ◽  
Author(s):  
Vijay Parashar ◽  
Chaitanya Aggarwal ◽  
Michael J. Federle ◽  
Matthew B. Neiditch
Keyword(s):  
Dna Binding ◽  
Structure Function ◽  
Cyclosporin A ◽  
Crystal Structures ◽  
Disulfide Bond ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
X Ray ◽  
Peptide Pheromone ◽  
Dimerization Interface

Peptide pheromone cell–cell signaling (quorum sensing) regulates the expression of diverse developmental phenotypes (including virulence) in Firmicutes, which includes common human pathogens, e.g.,Streptococcus pyogenesandStreptococcus pneumoniae. Cytoplasmic transcription factors known as “Rgg proteins” are peptide pheromone receptors ubiquitous in Firmicutes. Here we present X-ray crystal structures of aStreptococcusRgg protein alone and in complex with a tight-binding signaling antagonist, the cyclic undecapeptide cyclosporin A. To our knowledge, these represent the first Rgg protein X-ray crystal structures. Based on the results of extensive structure–function analysis, we reveal the peptide pheromone-binding site and the mechanism by which cyclosporin A inhibits activation of the peptide pheromone receptor. Guided by the Rgg–cyclosporin A complex structure, we predicted that the nonimmunosuppressive cyclosporin A analog valspodar would inhibit Rgg activation. Indeed, we found that, like cyclosporin A, valspodar inhibits peptide pheromone activation of conserved Rgg proteins in medically relevantStreptococcusspecies. Finally, the crystal structures presented here revealed that the Rgg protein DNA-binding domains are covalently linked across their dimerization interface by a disulfide bond formed by a highly conserved cysteine. The DNA-binding domain dimerization interface observed in our structures is essentially identical to the interfaces previously described for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface appears to be unique. We hypothesize that this disulfide bond may, under the right conditions, affect Rgg monomer–dimer equilibrium, stabilize Rgg conformation, or serve as a redox-sensitive switch.

scholarly journals Studies on crystal structures, active-centre geometry and depurinating mechanism of two ribosome-inactivating proteins

1995 ◽  
Vol 309 (1) ◽  
pp. 285-298 ◽  
Author(s):  
Q Huang ◽  
S Liu ◽  
Y Tang ◽  
S Jin ◽  
Y Wang
Keyword(s):  
Three Dimensional ◽  
Active Centre ◽  
Ribosome Inactivating Proteins ◽  
Aromatic Stacking ◽  
Replacement Method ◽  
Isomorphous Replacement ◽  
Molecular Replacement Method ◽  
The Difference ◽  
Related Proteins ◽  
The Relationship

Two ribosome-inactivating proteins, trichosanthin and alpha-momorcharin, have been studied in the forms of complexes with ATP or formycin, by an X-ray-crystallographic method at 1.6-2.0 A (0.16-0.20 nm) resolution. The native alpha-momorcharin had been studied at 2.2 A resolution. Structures of trichosanthin were determined by a multiple isomorphous replacement method. Structures of alpha-momorcharin were determined by a molecular replacement method using refined trichosanthin as the searching model. Small ligands in all these complexes have been recognized and built on the difference in electron density. All these structures have been refined to achieve good results, both in terms of crystallography and of ideal geometry. These two proteins show considerable similarity in their three-dimensional folding and to that of related proteins. On the basis of these structures, detailed geometries of the active centres of these two proteins are described and are compared with those of related proteins. In all complexes the interactions between ligand atoms and protein atoms, including hydrophobic forces, aromatic stacking interactions and hydrogen bonds, are found to be specific towards the adenine base. The relationship between the sequence conservation of ribosome-inactivating proteins and their active-centre geometry was analysed. A depurinating mechanism of ribosome-inactivating proteins is proposed on the basis of these results. The N-7 atom of the substrate base group is proposed to be protonated by an acidic residue in the active centre.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of (S)-3-hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum

2014 ◽  
Vol 70 (4) ◽  
pp. 485-488
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Crystal Volume

(S)-3-Hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum(CbHBD) is an enzyme that catalyzes the second step in the biosynthesis ofn-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. TheCbHBD protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 2 Mammonium sulfate, 0.1 MCAPS pH 10.5, 0.2 Mlithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space groupR3, with unit-cell parametersa=b= 148.5,c= 201.6 Å. With four molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 3.52 Å3 Da−1, which corresponds to a solvent content of approximately 65.04%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Low Bone Mass and Osteoporosis in Women Referring themselves to Dual X-Ray Absorptiometry - Experience with a Health Promotion Action

2010 ◽  
Vol 3 (1) ◽  
pp. 29-33
Author(s):  
Gerhard W. Goerres ◽  
Jaap Swanenburg ◽  
Daniel Uebelhart
Keyword(s):  
Health Promotion ◽  
Bone Mass ◽  
The Self ◽  
Low Bone Mass ◽  
X Ray ◽  
Female Patients ◽  
Bmd Testing ◽  
Significant Difference ◽  
The Difference ◽  
Prevalence Of Osteoporosis

Aims: This retrospective analysis was done to describe the difference in the prevalence of osteoporosis/low bone mass between women referring themselves to bone mineral density (BMD) testing with dual X-ray absorptiometry (DXA) and women referred by their family practitioner. Methods: Women were recruited by a health promotion action in a Swiss weekly periodical and compared with female patients sent by their physician for DXA testing for various medical indications during the same period. Patients under steroid treatment, known previous fracture and undergoing follow-up for low bone mass were excluded. Self referred women were compared to female patients aged 40 years and older and the same evaluation was repeated for women aged 65 and older. Results: No differences were found in the prevalence of osteoporosis /low bone mass in women referred by their physician vs those who were self referred. However, a significant difference was found with age: the self referred women were 63.1 ± 8.6 years of age whereas the patient group’s mean age was 59.7 ± 9.4 (p=0.0001, 95% CI of the difference: 21 – 61 years). Conclusion: We suggest that health promotion actions might be able to recruit the correct candidates for BMD testing, since we found no significant difference in the prevalence of osteoporosis/ low bone mass between self referred and physician referred women. Our data further suggest that physicians may react earlier on their patient’s risk profiles than the time frame of action by the self-referred women.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA fromRalstonia eutropha

2014 ◽  
Vol 70 (11) ◽  
pp. 1566-1569
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Ralstonia Eutropha ◽  
Condensation Reaction ◽  
Monomethyl Ether ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA fromRalstonia eutropha(RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA.RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 MTris–HCl pH 8.5 and 0.2 MtrimethylamineN-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space groupP21, with unit-cell parametersa= 68.38,b= 105.47,c= 106.91 Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.3 Å3 Da−1, which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Objective Quantification of Trochlea Dysplasia: Assessment of Morphology Difference Between Control and Chronic Patellofemoral Instability Patients

2017 ◽  
Vol 5 (4_suppl4) ◽  
pp. 2325967117S0013
Author(s):  
Andreas Voss ◽  
Andrea Achtnich ◽  
Shin Sanghin ◽  
Akin M. Murakami ◽  
Cory Edgar
Keyword(s):  
Trochlear Dysplasia ◽  
Patellofemoral Instability ◽  
Volumetric Analysis ◽  
Patella Instability ◽  
Control Cohort ◽  
X Ray ◽  
Significant Difference ◽  
Trochlea Dysplasia ◽  
The Difference ◽  
Gender Based

Aims and Objectives: Trochlear dysplasia is an important risk factor associated with patellofemoral instability, but it remains difficult to classify with consistency. Currently there is no objective way to quantify the dysplasia. The purpose of this study to define and quantify objectively the trochlea morphology by volume and length via computed tomography (CT). Hypothesis: A significant difference in trochlea groove volume and length is present within a cohort of patients with recurrent patellofemoral instability if compared to a control cohort of similar patients. Materials and Methods: One-hundred control patients (136 knees) were retrospectively reviewed and compared to 36 consecutive patients (72 knees) who were treated surgically for recurrent patella instability and known trochlea dysplasia based on a lateral x-ray. Trochlea morphology was analyzed from a pre-operative CT and data presented as trochlear sulcus volume trochlea length. To determine where along the trochlea length dysplasia is most variable, the trochlea length was radiographically divided into thirds, volume was quantified along that section and compared to control trochlea. Results: A significant difference in trochlea morphology exists between cohorts, volume (1.98 vs 3.77 cm3) and length (31.97 vs 34.66 mm). However, there appears to be a gender based difference in trochlea morphology. The trochlea volumetric analysis between the female cohorts (L: 2.02 cm3 vs. 2.94 cm3, R: 1.95 cm3 vs. 2.93 cm3) demonstrated significant less volume in instability patients (p<0.001). The proximal 30% of trochlea contributed the majority of dysplasia difference determined by comparing mean trochlea volume, 95% of the difference. This difference decreased in distal sections, 53% and 32% respectively. The total trochlea length did not appear to be significant (L: p=0.858, R: p=0.913). It appears dysplasia alone may not directly lead to symptoms demonstrated by trochlea volumetric comparisons within symptomatic recurrent patella instability and contralateral asymptomatic patella (p=0.274). Conclusion: The authors believe this reproducible technique can be used to quantify the trochlea morphology into measurements to be used describing the severity of the dysplasia. The data confirms that symptomatic trochlea dysplasia is a “proximal” process affecting early knee flexion contact between patella and trochlea.

scholarly journals Sample preparation, crystallization and structure solution of HisC fromMycobacterium tuberculosis

2013 ◽  
Vol 69 (4) ◽  
pp. 445-448 ◽  
Author(s):  
Nazia Nasir ◽  
Rajan Vyas ◽  
Bichitra K. Biswal
Keyword(s):  
Gel Filtration ◽  
Nitrilotriacetic Acid ◽  
Monomethyl Ether ◽  
X Ray Diffraction ◽  
X Ray ◽  
Metal Affinity ◽  
Replacement Method ◽  
Diffusion Technique ◽  
Structure Solution ◽  
Molecular Replacement Method

Histidinolphosphate aminotransferase (HisC; Rv1600) fromMycobacterium tuberculosiswas overexpressed inM. smegmatisand purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space groupP3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using itsCorynebacterium glutamicumcounterpart as a search model. HisC is a dimer in the crystal as well as in solution.

scholarly journals Radiographic analysis of Hallux valgus. A study on foot arch by severity

2017 ◽  
Vol 2 (3) ◽  
pp. 2473011417S0003
Author(s):  
Yamada Takahiro ◽  
Norio Usami ◽  
Ikezawa Hiroko ◽  
Naoko Takatori ◽  
Eiichi Hiraishi ◽  
...  
Keyword(s):  
Hallux Valgus ◽  
Bone Structure ◽  
Sagittal Plane ◽  
Severe Case ◽  
Mild Case ◽  
X Ray ◽  
Significant Difference ◽  
Foot Arch ◽  
Group A ◽  
The Difference

Category: Bunion Introduction/Purpose: X-ray measurement for hallux valgus has been conducted with various results in the evaluation. However, it was not quite clarified yet and it still remains questionable why a mild case shifts to a severe case during the course. We report about the difference between the severity and the foot shape. Methods: The study subjects were 206 feet of 138 patients. Mild case: 80 feet, mean age 53 y.o (A) Moderate case: 61 feet, 62 y.o (B) Severe case: 65 feet, 67 y.o (C) For the examination items, HVA, M1-2 angle, and M1-5 angle were measured with the x-ray frontal radiograph for loading position, and First, Second, Fifth intermetatarsal angle (M1Y/M2Y/M5Y) on sagittal plane were also measured. We also evaluated the foot arch ratio with Yokokura Method, then compared/examined those results after dividing the cases into the mild, moderate, and severe group with age bracket. Results: The age of Group C were older than Group A, B. M1-2 angle: In younger generation, three is significant difference between Group A and B. M1-5 angle: Significant in 60 s between A and B. In 70 s, significant: all group. Navicular height: all group is low arch with aging. Significant between A and C It is becoming low arch at Lisfranc level with aging in all group. There is significantly low height at M5 with aging in all group. On sagittal plane, bone axis of M1 and M2 is lowered at 40 s in Group A and B and 70 s in Group C. Conclusion: In hallux valgus, the foot shape was changed in coronal and sagittal plane. It means the collapse of bone structure at foot and ankle. It may possibly be shifting to a severe case with aging. However, we could not find any result definitely suggesting such condition.

scholarly journals Endoscopist’s occupational dose evaluation related to correct wearing of dosimeter during X-ray-guided procedures

2019 ◽  
Vol 07 (03) ◽  
pp. E367-E371 ◽  
Author(s):  
Roberta Gerasia ◽  
Dario Ligresti ◽  
Fabio Cipolletta ◽  
Antonino Granata ◽  
Ilaria Tarantino ◽  
...  
Keyword(s):  
Radiation Exposure ◽  
Radiation Source ◽  
Whole Body ◽  
Back Side ◽  
Correct Evaluation ◽  
X Ray ◽  
Dose Evaluation ◽  
Occupational Dose ◽  
Significant Difference ◽  
The Difference

Abstract Background Since endoscopists performing procedures in the endoscopy suite can change their position by turning their back, side or front toward the X-ray source, this study aimed to establish whether dosimeter position affects the correct evaluation of an endoscopist’s personal radiation exposure during X-ray-guided procedures. Materials and methods Between January and February 2018, two dosimeters specularly placed outside the lead apron (anterior one on the chest and posterior one on the back) measured endoscopists’ personal dose equivalent (Hp) during 62 X-ray-guided procedures on adult and pediatric patients. Procedures were divided into three groups considering the position taken by the endoscopist with respect to the radiation source. For each group, the difference between mean Hp from the anterior and posterior dosimeters was calculated. Results A statistically significant difference in mean Hp was recorded for the endoscopists’ frontal and back positions (P = 0.014, and P < 0.00001, respectively). No significant difference was found in mean Hp for the side position (P = 0.489). Conclusions The position of personal dosimeters affects the correct evaluation of endoscopists’ radiation exposure during X-ray-guided procedures when frontal and back positions were recorded. To correctly evaluate radiation doses, the whole-body dosimeter should be worn according to the position of the endoscopist with respect to the radiation source; otherwise, it results in an incorrect personal dose evaluation, which may lead to substantial underestimation of staff exposure.

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