Potential use of ultrasound to promote protein crystallization

2010 ◽  
Vol 43 (6) ◽  
pp. 1419-1425 ◽  
Author(s):  
Rosa Crespo ◽  
Pedro M. Martins ◽  
Luís Gales ◽  
Fernando Rocha ◽  
Ana M. Damas
Keyword(s):  
Protein Crystallization ◽  
Crystal Formation ◽  
Data Sets ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
Promoting Effect ◽  
X Ray ◽  
X Ray Crystallography ◽  
Egg White Lysozyme ◽  
Crystallization Experiments

This work shows promising applications of ultrasound in promoting protein crystallization, which is important for structure determination by X-ray crystallography. It was observed that ultrasound can be used as a nucleation promoter as it decreases the energy barrier for crystal formation. Crystallization experiments on egg-white lysozyme were carried out with and without ultrasonic irradiation using commercial crystallization plates placed in temperature-controlled water baths. The nucleation-promoting effect introduced by ultrasound is illustrated by the reduction of the metastable zone width, as measured by the isothermal microbatch technique. The same effect was confirmed by the increased number of conditions leading to the formation of crystals when vapour diffusion techniques were carried out in the presence of ultrasound. By inducing faster nucleation, ultrasound leads to protein crystals grown at low supersaturation levels, which are known to have better diffraction properties. In fact, X-ray diffraction data sets collected using 13 lysozyme crystals (seven grown with ultrasound and six without) show an average 0.1 Å improvement in the resolution limit when ultrasound was used (p< 0.10). Besides the immediate application of ultrasound in nucleation promotion, the preliminary diffraction results also suggest a promising application in crystal quality enhancement.

scholarly journals Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol

IUCrJ ◽  
2015 ◽  
Vol 2 (5) ◽  
pp. 534-544 ◽  
Author(s):  
S. Fili ◽  
A. Valmas ◽  
M. Norrman ◽  
G. Schluckebier ◽  
D. Beckers ◽  
...  
Keyword(s):  
Human Insulin ◽  
Organic Ligand ◽  
European Synchrotron Radiation Facility ◽  
X Ray Diffraction ◽  
Ph Variation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Swiss Light Source ◽  
Crystallization Experiments ◽  
Ph Range

This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50–8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

scholarly journals A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

2003 ◽  
Vol 36 (2) ◽  
pp. 315-318 ◽  
Author(s):  
J. Brown ◽  
T. S. Walter ◽  
L. Carter ◽  
N. G. A. Abrescia ◽  
A. R. Aricescu ◽  
...  
Keyword(s):  
Scale Up ◽  
Data Sets ◽  
Molecular Replacement ◽  
Research Groups ◽  
X Ray Diffraction ◽  
X Ray ◽  
Crystallization Experiments ◽  
Crystallization Conditions ◽  
Synchrotron Beamlines

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of X-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full X-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

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.

scholarly journals Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 500-508 ◽  
Author(s):  
Emma V. Beale ◽  
Anna J. Warren ◽  
José Trincão ◽  
James Beilsten-Edmands ◽  
Adam D. Crawshaw ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Protein Complexes ◽  
Protein Crystals ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
X Ray ◽  
X Ray Crystallography ◽  
Significant Difference ◽  
Scanning Electron

Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

Crystallization and preliminary X-ray diffraction studies on the conserved GTPase domain of the signal recognition particle from Acidianus ambivalens

1999 ◽  
Vol 55 (11) ◽  
pp. 1949-1951 ◽  
Author(s):  
Guillermo Montoya ◽  
Kai te Kaat ◽  
Ralf Moll ◽  
Günter Schäfer ◽  
Irmgard Sinning
Keyword(s):  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Ras Gtpases ◽  
Crystallization Experiments

The signal recognition particle (SRP) of bacteria consists of only one protein, known as Ffh or the SRP54 homologue, which forms a complex with 4.5S RNA. It also binds to signal peptides and contains a GTPase which displays interesting differences to Ras GTPases. The conserved NG-domain of Ffh from the archaebacterium Acidianus ambivalens was cloned and overexpressed with a C-terminal His tag in Escherichia coli. Crystallization experiments of the native protein as well as of the Thr112Ala mutant, which is deficient in GTP hydrolysis, resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 64.5, b = 128.3, c = 72.0 Å. At cryogenic temperatures, the crystals diffracted to a resolution limit of 2.8 Å using a rotating-anode generator and contain one molecule per asymmetric unit. A native data set has been collected using synchrotron radiation to around 2.0 Å resolution. Selenomethionine protein was produced; its crystals diffract in-house to about 2.8 Å resolution.

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 Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

2019 ◽  
Vol 75 (10) ◽  
pp. 937-946 ◽  
Author(s):  
Rebecka Andersson ◽  
Cecilia Safari ◽  
Petra Båth ◽  
Robert Bosman ◽  
Anastasya Shilova ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Structures ◽  
Cubic Phase ◽  
Crystal Formation ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Lipidic Cubic Phase ◽  
Serial Crystallography

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

Synthesis and characterization of a linked, π-π stacked organotin compound of 2-mercapto-6-nitrobenzothiazolyl diphenyltin chloride with 2-ethoxyl-6-nitrobenzothiazole

2003 ◽  
Vol 81 (7) ◽  
pp. 825-831 ◽  
Author(s):  
Chunlin Ma ◽  
Qin Jiang ◽  
Rufen Zhang
Keyword(s):  
Title Compound ◽  
Organotin Compound ◽  
X Ray Diffraction ◽  
X Ray ◽  
Stacking Interaction ◽  
X Ray Crystallography ◽  
H Nmr ◽  
Π Stacking ◽  
Π Stacking Interaction ◽  
Molecular Components

The new organotin compound, Ph2Sn(Cl)[S(C7H3N2O2S)]·[(C7H3N2O2S)OEt], assembled by an intermolecular aromatic benzothiazole–benzothiazole π-π stacking interaction, has been synthesized by the reaction of diphenyltin dichloride with 2-mercapto-6-nitrobenzothiazole. The title compound was characterized by elemental, IR, 1H NMR, and X-ray crystallography analyses. Single-crystal X-ray diffraction data reveals that the title compound has two different molecular components. The component Ph2Sn(Cl)[S(C7H3N2O2S)] has a pentacoordinate tin, which further forms an infinite one-dimensional chain by intermolecular non-bonded Cl···S interactions, resulting in an intercalation lattice that holds (C7H3N2O2S)OEt molecules. The formation of the molecule (C7H3N2O2S)OEt as well as its intercalated mechanism has also been discussed.Key words: organotin, assemble, π-π stacking interaction, 2-mercapto-6-nitrobenzothiazole, non-bonded interaction, crystal structure.

scholarly journals Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

2017 ◽  
Vol 73 (11) ◽  
pp. 607-611
Author(s):  
Fang Lu ◽  
Bei Zhang ◽  
Yong Liu ◽  
Ying Song ◽  
Gangxing Guo ◽  
...  
Keyword(s):  
Unit Cell ◽  
Diffusion Method ◽  
Data Sets ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

Phytases are phosphatases that hydrolyze phytates to less phosphorylatedmyo-inositol derivatives and inorganic phosphate. β-Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β-propeller phytase fromBacillussp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vapour-diffusion method in a condition consisting of 0.2 Msodium chloride, 0.1 MTris pH 8.5, 25%(w/v) PEG 3350 at 289 K. X-ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH-DI crystals belonged to space groupC121, with unit-cell parametersa = 156.84,b = 45.54,c = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH-DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1and a solvent content of 43.26%. Crystals of selenomethionine-substituted PhyH-DI belonged to space groupC2221, with unit-cell parametersa = 94.71,b= 97.03,c= 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1and a solvent content of 49.64%. Initial phases for PhyH-DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH-DI.

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