Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

2015 ◽  
Vol 71 (5) ◽  
pp. 1095-1101 ◽  
Author(s):  
Manuela Gorgel ◽  
Andreas Bøggild ◽  
Jakob Jensen Ulstrup ◽  
Manfred S. Weiss ◽  
Uwe Müller ◽  
...  
Keyword(s):  
Protein Structure ◽  
Sodium Ion ◽  
Data Sets ◽  
Threshold Values ◽  
X Ray Diffraction ◽  
X Ray ◽  
Single Well ◽  
Anomalous Signal ◽  
Pilin Protein

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicatorRanom/Rp.i.m., an inconsistency in the documentation of the anomalousRfactor is noted and reported.

scholarly journals SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

2019 ◽  
Vol 75 (12) ◽  
pp. 731-737 ◽  
Author(s):  
Abhin Kumar Megta ◽  
Airi Palva ◽  
Ingemar von Ossowski ◽  
Vengadesan Krishnan
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Crystal Form ◽  
Data Sets ◽  
Structural Factors ◽  
Lysine Methylation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Dual Functional ◽  
Anomalous Signal

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6522 or P6122, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = β = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

Determination of depth profiles from X-ray diffraction data

1993 ◽  
Vol 8 (2) ◽  
pp. 122-126 ◽  
Author(s):  
Paul Predecki
Keyword(s):  
Diffraction Data ◽  
Direct Method ◽  
Sample Surface ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Data Set ◽  
X Ray ◽  
Depth Profiles ◽  
A Direct Method

A direct method is described for determining depth profiles (z-profiles) of diffraction data from experimentally determined τ-profiles, where z is the depth beneath the sample surface and τ is the 1/e penetration depth of the X-ray beam. With certain assumptions, the relation between these two profile functions can be expressed in the form of a Laplace transform. The criteria for fitting experimental τ-data to functions which can be utilized by the method are described. The method was applied to two τ-data sets taken from the literature: (1) of residual strain in an A1 thin film and (2) of residual stress in a surface ground A12O3/5vol% TiC composite. For each data set, it was found that the z-profiles obtained were of two types: oscillatory and nonoscillatory. The nonoscillatory profiles appeared to be qualitatively consistent for a given data set. The oscillatory profiles were considered to be not physically realistic. For the data sets considered, the nonoscillatory z-profiles were found to lie consistently above the corresponding τ-profiles, and to approach the τ-profiles at large z, as expected from the relation between the two.

The application of the molecular replacement method to the de novo determination of protein structure

1991 ◽  
Vol 24 (4) ◽  
pp. 399-424 ◽  
Author(s):  
Michael C. Lawrence
Keyword(s):  
Protein Structure ◽  
De Novo ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Electron Density Map ◽  
Novel Protein

The determination of a novel protein structure by X-ray diffraction is seldom straightforward. Three hurdles present themselves (i) the protein must be purified in sufficient quantity to allow crystallization trials, (ii) crystals must be grown to adequate size and must diffract to a resolution that will allow atomic detail to be revealed, and (iii) phases must be determined for the diffracted X-ray beams in order that an initial electron-density map may be calculated.

Use of Image-Processing Tools for Texture Analysis of High-Energy X-ray Synchrotron Data

1998 ◽  
Vol 31 (5) ◽  
pp. 647-653 ◽  
Author(s):  
R. Fisker ◽  
H. F. Poulsen ◽  
J. Schou ◽  
J. M. Carstensen ◽  
S. Garbe
Keyword(s):  
Image Processing ◽  
Nonlinear Least Squares ◽  
High Energy ◽  
Three Dimensions ◽  
Polycrystalline Materials ◽  
Data Sets ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray

The introduction of synchrotron beamlines for high-energy X-ray diffraction raises new possibilities for texture determination of polycrystalline materials. The local texture can be mapped out in three dimensions and texture developments can be studiedin situin complicated environments. However, it is found that a full alignment of the two-dimensional detector used in many cases is impractical and that data-sets are often partially subject to geometric restrictions. Estimating the parameters of the traces of the Debye–Scherrer cones on the detector therefore becomes a concern. Moreover, the background may vary substantially on a local scale as a result of inhomogeneities in the sample environmentetc. A set of image-processing tools has been employed to overcome these complications. An automatic procedure for estimating the parameters of the traces (taken as ellipses) is described, based on a combination of a circular Hough transform and nonlinear least-squares fitting. Using the estimated ellipses the background is subtracted and the intensity along the Debye–Scherrer cones is integrated by a combined fit of the local diffraction pattern. The corresponding algorithms are presented together with the necessary coordinate transform for pole-figure determination. The image-processing tools may be useful for the analysis of noisy or partial powder diffraction data-sets in general, provided flat two-dimensional detectors are used.

scholarly journals Determination of Patterson group symmetry from sparse multi-crystal data sets in the presence of an indexing ambiguity

2018 ◽  
Vol 74 (5) ◽  
pp. 405-410 ◽  
Author(s):  
Richard J. Gildea ◽  
Graeme Winter
Keyword(s):  
Polar Space ◽  
Group Symmetry ◽  
Data Sets ◽  
X Ray Diffraction ◽  
X Ray ◽  
Space Groups ◽  
Partial Data ◽  
Multiple Samples

Combining X-ray diffraction data from multiple samples requires determination of the symmetry and resolution of any indexing ambiguity. For the partial data sets typical of in situ room-temperature experiments, determination of the correct symmetry is often not straightforward. The potential for indexing ambiguity in polar space groups is also an issue, although methods to resolve this are available if the true symmetry is known. Here, a method is presented to simultaneously resolve the determination of the Patterson symmetry and the indexing ambiguity for partial data sets.

Sem and X-Ray Analysis of Renal and Biliary Calculi

1976 ◽  
Vol 34 ◽  
pp. 306-307
Author(s):  
R. J. Narconis ◽  
G. L. Johnson
Keyword(s):  
Chemical Constituents ◽  
Natural State ◽  
X Ray Diffraction ◽  
Chemical Methods ◽  
X Ray ◽  
Additional Dimension ◽  
Biliary Calculi ◽  
Calculus Formation ◽  
Scanning Electron

Analysis of the constituents of renal and biliary calculi may be of help in the management of patients with calculous disease. Several methods of analysis are available for identifying these constituents. Most common are chemical methods, optical crystallography, x-ray diffraction, and infrared spectroscopy. The application of a SEM with x-ray analysis capabilities should be considered as an additional alternative.A scanning electron microscope equipped with an x-ray “mapping” attachment offers an additional dimension in its ability to locate elemental constituents geographically, and thus, provide a clue in determination of possible metabolic etiology in calculus formation. The ability of this method to give an undisturbed view of adjacent layers of elements in their natural state is of advantage in determining the sequence of formation of subsequent layers of chemical constituents.

Defect-Mediated Conductivity Enhancements in Na3-xPn1-xWxS4 (Pn = P, Sb) using Aliovalent Substitutions

2019 ◽  
Author(s):  
Till Fuchs ◽  
Sean Culver ◽  
Paul Till ◽  
Wolfgang Zeier
Keyword(s):  
Ionic Conductivity ◽  
Vacancy Concentration ◽  
Sodium Ion ◽  
High Ionic Conductivity ◽  
Ionic Conductors ◽  
X Ray Diffraction ◽  
X Ray ◽  
Solid State Batteries ◽  
Ion Conducting ◽  
Very High

<p>The sodium-ion conducting family of Na<sub>3</sub><i>Pn</i>S<sub>4</sub>, with <i>Pn</i> = P, Sb, have gained interest for the use in solid-state batteries due to their high ionic conductivity. However, significant improvements to the conductivity have been hampered by the lack of aliovalent dopants that can introduce vacancies into the structure. Inspired by the need for vacancy introduction into Na<sub>3</sub><i>Pn</i>S<sub>4</sub>, the solid solutions with WS<sub>4</sub><sup>2-</sup> introduction are explored. The influence of the substitution with WS<sub>4</sub><sup>2-</sup> for PS<sub>4</sub><sup>3-</sup> and SbS<sub>4</sub><sup>3-</sup>, respectively, is monitored using a combination of X-ray diffraction, Raman and impedance spectroscopy. With increasing vacancy concentration improvements resulting in a very high ionic conductivity of 13 ± 3 mS·cm<sup>-1</sup> for Na<sub>2.9</sub>P<sub>0.9</sub>W<sub>0.1</sub>S<sub>4</sub> and 41 ± 8 mS·cm<sup>-1</sup> for Na<sub>2.9</sub>Sb<sub>0.9</sub>W<sub>0.1</sub>S<sub>4</sub> can be observed. This work acts as a stepping-stone towards further engineering of ionic conductors using vacancy-injection via aliovalent substituents.</p>

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