Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection?

2009 ◽  
Vol 16 (2) ◽  
pp. 191-204 ◽  
Author(s):  
Sofia Macedo ◽  
Maria Pechlaner ◽  
Walther Schmid ◽  
Martin Weik ◽  
Katsuko Sato ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Active Sites ◽  
Structural Data ◽  
Potassium Hexacyanoferrate ◽  
X Ray Diffraction ◽  
Metal Centre ◽  
Data Set ◽  
Synchrotron Source ◽  
X Ray ◽  
X Ray Crystallography

One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV–Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV–Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.

scholarly journals Practical Aspects of the SAMPL Challenge: Providing an Extensive Experimental Data Set for the Modeling Community

2009 ◽  
Vol 14 (10) ◽  
pp. 1245-1250 ◽  
Author(s):  
Janet Newman ◽  
Vincent J. Fazio ◽  
Tom T. Caradoc-Davies ◽  
Kim Branson ◽  
Thomas S. Peat
Keyword(s):  
Evaluation Study ◽  
X Ray Diffraction ◽  
Data Set ◽  
X Ray ◽  
Software Application ◽  
X Ray Crystallography ◽  
Software Packages ◽  
Structure Solution ◽  
Fragment Library ◽  
Extensive Experimental Data

To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if combination of fragments is an appropriate strategy. A further set of data was obtained from co-crystallizing the pooled fragments with the protein. X-ray diffraction data were collected on approximately 1000 crystals at the Australian Synchrotron, and these data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of effector protein MoHrip2 fromMagnaporthe oryzae

2013 ◽  
Vol 69 (4) ◽  
pp. 463-467 ◽  
Author(s):  
Mengjie Liu ◽  
Xinqi Liu ◽  
Hongmei Zeng ◽  
Dewen Qiu
Keyword(s):  
Magnaporthe Oryzae ◽  
Pathogenic Fungus ◽  
Diffusion Method ◽  
Effector Protein ◽  
X Ray Diffraction ◽  
Data Set ◽  
Synchrotron Source ◽  
X Ray ◽  
Vapour Diffusion ◽  
Anomalous Signal

MoHrip2, a novel effector protein from the pathogenic fungusMagnaporthe oryzae, was purified and crystallized using the sitting-drop vapour-diffusion method. Native crystals and selenomethionine-labelled crystals were obtained using 2.2 Mammonium sulfate as a precipitant. A native data set was collected to 2.0 Å resolution at 100 K using an in-house X-ray source and a selenomethionine-labelled data set containing anomalous signal was collected to 1.8 Å resolution at 100 K using a synchrotron source. Based on the anomalous signal generated from the Se atom, the MoHrip2 structure was successfully solved using the single-wavelength anomalous dispersion (SAD) method.

scholarly journals Supplemental Material: Syncing fault rock clocks: Direct comparison of U-Pb carbonate and K-Ar illite fault dating methods

2020 ◽  
Author(s):  
Catherine Mottram ◽  
et al.
Keyword(s):  
Structural Data ◽  
X Ray Diffraction ◽  
Fault Rock ◽  
Data Set ◽  
X Ray ◽  
Dating Methods ◽  
Carbonate Sample

Full analytical materials, gouge and detailed carbonate sample descriptions and locations, summary of additional U-Pb geochronology plots and interpretation, structural data, and Data Set S1 (K-Ar results), Data Set S2 (X-ray diffraction results), Data Set S3 (U-Pb carbonate results), and Data Set S4 (U-Pb standard reproducibility). <br>

Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius

1999 ◽  
Vol 55 (7) ◽  
pp. 1348-1349 ◽  
Author(s):  
Giuseppina De Simone ◽  
Giuseppe Manco ◽  
Stefania Galdiero ◽  
Angela Lombardi ◽  
Mosè Rossi ◽  
...  
Keyword(s):  
Ammonium Sulfate ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Synchrotron Source ◽  
X Ray ◽  
Cell Parameters ◽  
Alicyclobacillus Acidocaldarius ◽  
First Time

EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time. Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106.4 Å. A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 Å resolution, using a single frozen crystal.

scholarly journals Supplemental Material: Syncing fault rock clocks: Direct comparison of U-Pb carbonate and K-Ar illite fault dating methods

2020 ◽  
Author(s):  
Catherine Mottram ◽  
et al.
Keyword(s):  
Structural Data ◽  
X Ray Diffraction ◽  
Fault Rock ◽  
Data Set ◽  
X Ray ◽  
Dating Methods ◽  
Carbonate Sample

Full analytical materials, gouge and detailed carbonate sample descriptions and locations, summary of additional U-Pb geochronology plots and interpretation, structural data, and Data Set S1 (K-Ar results), Data Set S2 (X-ray diffraction results), Data Set S3 (U-Pb carbonate results), and Data Set S4 (U-Pb standard reproducibility). <br>

scholarly journals Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction

IUCrJ ◽  
2014 ◽  
Vol 1 (5) ◽  
pp. 349-360 ◽  
Author(s):  
Michael Heymann ◽  
Achini Opthalage ◽  
Jennifer L. Wierman ◽  
Sathish Akella ◽  
Doletha M. E. Szebenyi ◽  
...  
Keyword(s):  
Room Temperature ◽  
Protein Crystallization ◽  
Glucose Isomerase ◽  
Feedback Mechanism ◽  
X Ray Diffraction ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography ◽  
Negative Feedback Mechanism ◽  
On Chip

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

Tubulin structure at intermediate resolution

1995 ◽  
Vol 53 ◽  
pp. 852-853
Author(s):  
K. H. Downing ◽  
S. G. Wolf ◽  
E. Nogales
Keyword(s):  
High Resolution ◽  
Structural Data ◽  
Therapeutic Drug ◽  
Zinc Ions ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Negative Stain ◽  
Functional Mechanisms ◽  
Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

Texture of submicron Ni-Mn-Ga films studied by X-ray diffraction at the ANKA synchrotron source

2007 ◽  
Vol 2007 (suppl_26) ◽  
pp. 229-234 ◽  
Author(s):  
V. A. Chernenko ◽  
S. Doyle ◽  
M. Kohl ◽  
P. Müllner ◽  
S. Besseghini ◽  
...  
Keyword(s):  
X Ray Diffraction ◽  
Synchrotron Source ◽  
X Ray

Role of the Structure and Electronic Properties of Fe2O3-MoO3 Catalyst on the Dehydration of Isopropyl Alcohol

1993 ◽  
Vol 58 (7) ◽  
pp. 1591-1599 ◽  
Author(s):  
Abd El-Aziz A. Said
Keyword(s):  
Active Sites ◽  
Isopropyl Alcohol ◽  
Oxide Catalyst ◽  
Flow System ◽  
Charge Carriers ◽  
X Ray Diffraction ◽  
X Ray ◽  
Catalyst Composition ◽  
Surface Active

Molybdenum oxide catalyst doped or mixed with (1 - 50) mole % Fe3+ ions were prepared. The structure of the original samples and the samples calcined at 400 °C were characterized using DTA, X-ray diffraction and IR spectra. Measurements of the electrical conductivity of calcined samples with and without isopropyl alcohol revealed that the conductance increases on increasing the content of Fe3+ ions up to 50 mole %. The activation energies of charge carriers were determined in presence and absence of the alcohol. The catalytic dehydration of isopropyl alcohol was carried out at 250 °C using a flow system. The results obtained showed that the doped or mixed catalysts are active and selective towards propene formation. However, the catalyst containing 40 mole % Fe3+ ions exhibited the highest activity and selectivity. Correlations were attempted to the catalyst composition with their electronic and catalytic properties. Probable mechanism for the dehydration process is proposed in terms of surface active sites.

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