scholarly journals A new view on crystal harvesting

2014 ◽  
Vol 47 (3) ◽  
pp. 1158-1161 ◽  
Author(s):  
Joseph R. Luft ◽  
Thomas D. Grant ◽  
Jennifer R. Wolfley ◽  
Edward H. Snell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
High Throughput Screening ◽  
Scale Up ◽  
Trna Synthetase ◽  
Objective Lens ◽  
X Ray ◽  
X Ray Crystallography ◽  
Automated Process ◽  
Structural Solution

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain ofSaccharomyces cerevisiaeglutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Reinhard Krämer ◽  
Christine Ziegler
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Protein Interactions ◽  
Regulation Mechanism ◽  
Protein Protein Interactions ◽  
Molecular Switching ◽  
X Ray ◽  
X Ray Crystallography ◽  
Terminal Domain ◽  
Domain Reorientation

Abstract Activation of the osmoregulated trimeric betaine transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly α-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.

Studies Directed Towards the Total Synthesis of Anticapsin. X-Ray Crystal-Structure of (1RS,2SR,4RS,5RS,6SR)-5-Hydroxy-2-phthalimido-1-trimethylsilyloxy-bicyclo[2.2.2]octane-2,6-carbolactone

1990 ◽  
Vol 43 (11) ◽  
pp. 1827 ◽  
Author(s):  
MJ Crossley ◽  
TW Hambley ◽  
AW Stamford
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Total Synthesis ◽  
Synthetic Approach ◽  
Hydrogen Atoms ◽  
Full Matrix ◽  
X Ray ◽  
X Ray Crystallography ◽  
Relative Stereochemistry ◽  
Atomic Parameters

The relative stereochemistry of methyl 2-phthalimido-1- trimethylsilyloxybicyclo[2.2.2]oct-5-ene-2-carboxylate (9) and its 5,6-epoxide (10), intermediates in a synthetic approach to the amino acid antibiotic anticapsin, were established by the TiCl4-mediated cyclization of (10) to the carbolactone (12); the structure of which was proved by single-crystal X-ray crystallography. Full-matrix least- squares refinement of all atomic parameters with individual isotropic thermal parameters for the hydrogen atoms by using 1446 reflections converged at R 0.036. Crystals of (12) are monoclinic, P21/c, a 12.342(3), b 12.239(2), c 13.405(3) Ǻ, β 99.34(2)°, Z 4.

scholarly journals A defined structural unit enables de novo design of small-molecule–binding proteins

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1227-1233 ◽  
Author(s):  
Nicholas F. Polizzi ◽  
William F. DeGrado
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Small Molecules ◽  
Structural Unit ◽  
De Novo ◽  
Computational Design ◽  
De Novo Design ◽  
X Ray ◽  
X Ray Crystallography ◽  
Chemical Groups

The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.

scholarly journals Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

2008 ◽  
Vol 105 (46) ◽  
pp. 17682-17687 ◽  
Author(s):  
Jessica Bockhorn ◽  
Bharvi Balar ◽  
Dongming He ◽  
Eden Seitomer ◽  
Paul R. Copeland ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Cost Effective ◽  
Anomalous Dispersion ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Cost Effective Method ◽  
Strain Collection

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

scholarly journals A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors

2017 ◽  
Vol 23 (1) ◽  
pp. 11-22
Author(s):  
Stephen A. St-Gallay ◽  
Neil Bennett ◽  
Susan E. Critchlow ◽  
Nicola Curtis ◽  
Gareth Davies ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Isothermal Calorimetry ◽  
Binding Mode ◽  
Carbonyl Oxygen ◽  
Enzyme Concentration ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Hill

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

scholarly journals Fragment-based screening by X-ray crystallography: an alternative to high throughput screening

2005 ◽  
Vol 61 (a1) ◽  
pp. c246-c246
Author(s):  
A. Cleasby ◽  
L. Devine ◽  
M. Frederickson ◽  
M. Hartshorn ◽  
I. Tickle ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Synthesis and Characterization of Various Amino Acid Derived Thiohydantoins

Proceedings ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 37
Author(s):  
Petar Stanić ◽  
Marija Živković ◽  
Biljana Šmit
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Allyl Isothiocyanate ◽  
Structural Diversity ◽  
Biologically Active ◽  
Reaction Conditions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sulfur Containing

Hydantoins and their sulfur containing analogues, thiohydantoins, are cyclic ureides that have attracted huge attention ever since their discovery. Most of them are biologically active compounds and several points of structural diversity have made them very synthetically attractive. Although substituents can be introduced to the hydantoin nucleus, most substituted hydantoins are synthesized from substrates already containing these groups, while forming the hydantoin nucleus. This is a common route to the synthesis of hydantoins and one of them is employed in this study. A series of 3-allyl-2-thiohydantoins is synthesized from various α-amino acids in a reaction with allyl isothiocyanate. The substitution of the acquired thiohydantoin depends on the structure of the starting α-amino acid. The residual group of the α-amino acid becomes the substituent at the C5-position, while N-monosubstituted amino acids give rise to a substituent in the N1-position. The reaction is carried out in a two-step process and the reaction conditions generally depend on the nature of the amino acid itself. All thiohydantoins are obtained in a good yield and fully characterized by NMR and IR spectroscopy, as well as X-ray crystallography.

Crystal structures of N6-modified-amino acid related nucleobase analogs (II): hybrid adenine-β-alanine and adenine-GABA molecules

2019 ◽  
Vol 43 (24) ◽  
pp. 9680-9688 ◽  
Author(s):  
Angel García-Raso ◽  
Angel Terrón ◽  
Adela López-Zafra ◽  
Andrés García-Viada ◽  
Agostina Barta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dft Calculations ◽  
Crystal Structures ◽  
X Ray ◽  
Π Interactions ◽  
X Ray Crystallography

H-Bonding networks and anion–π interactions in the crystal structures of N6-modified-amino acid adenine analogs are investigated using X-ray crystallography and DFT calculations.

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