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.

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

IUCrJ ◽  
2015 ◽  
Vol 2 (5) ◽  
pp. 601-601 ◽  
Author(s):  
Michael Heymann ◽  
Achini Opathalage ◽  
Jennifer L. Wierman ◽  
Sathish Akella ◽  
Doletha M. E. Szebenyi ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Room Temperature ◽  
Protein Crystallization ◽  
X Ray Diffraction ◽  
X Ray ◽  
Serial Crystallography ◽  
On Chip

The name of one of the authors in the article by Heymannet al.[(2014),IUCrJ,1, 349–360] is corrected.

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 Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase

2015 ◽  
Vol 71 (11) ◽  
pp. 1448-1452 ◽  
Author(s):  
John-Paul Bacik ◽  
Sophanit Mekasha ◽  
Zarah Forsberg ◽  
Andrey Kovalevsky ◽  
Jay C. Nix ◽  
...  
Keyword(s):  
High Resolution ◽  
Diffraction Data ◽  
Room Temperature ◽  
X Ray Diffraction ◽  
Lytic Polysaccharide Monooxygenase ◽  
Data Set ◽  
X Ray ◽  
Processing Enzymes ◽  
Bacteria And Fungi ◽  
Polysaccharide Monooxygenase

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1–3 mm3) of a chitin-processing LPMO from the Gram-positive soil bacteriumJonesia denitrificanswere grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space groupP212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.

X-Ray Diffraction Study of the Electron Density and Anharmonicity in K2PtCl6

1993 ◽  
Vol 48 (1-2) ◽  
pp. 12-20 ◽  
Author(s):  
Renzo Restori ◽  
Dieter Schwarzenbach
Keyword(s):  
Charge Density ◽  
Room Temperature ◽  
Heavy Atom ◽  
X Ray Diffraction ◽  
Large Core ◽  
Electron Densities ◽  
Core Electron ◽  
Data Set ◽  
X Ray ◽  
Atomic Displacements

Abstract X-ray diffraction data in heavy-atom compounds may be sensitive to anharmonic atomic displacements, since the large core electron densities result in appreciable scattering amplitudes at large reciprocal distances. Since bonding electron densities may also exhibit sharp features affecting high-order reflections, they may be difficult to distinguish from anharmonic effects. We have analyzed an accurate room-temperature single-crystal X-ray data set of K2 PtCl6 using least-squares anharmonic displacement and charge density formalisms. The Hirshfeld charge density formalism, which has successfully been applied to many light-atom structures, fails to parametrize satisfactorily the data, whereas the electron densities at K and CI are easily accounted for by an anharmonic Gram-Charlier expansion to 4th order. Densities around Pt are parametrized only by a combination of anharmonicity and charge density formalisms. If economical parametrizations of the experimental data are preferred to more complicated ones, anharmonicity may be conjectured to play an important rôle while the main bonding feature consists of a preferential occupation of the 5d-orbitals of Pt with t2g symmetry.

Combining `dry' co-crystallization andin situdiffraction to facilitate ligand screening by X-ray crystallography

2015 ◽  
Vol 71 (8) ◽  
pp. 1777-1787 ◽  
Author(s):  
Muriel Gelin ◽  
Vanessa Delfosse ◽  
Frédéric Allemand ◽  
François Hoh ◽  
Yoann Sallaz-Damaz ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
X Ray Diffraction ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ligand Screening ◽  
Alternative Approach ◽  
Manual Handling ◽  
Stock Solutions ◽  
Established Technique

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps – soaking crystals with ligand and the subsequent harvesting – are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly `in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.

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.

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