Initiating Heavy-atom Based Phasing by Multi-Dimensional Molecular Replacement

To obtain an electron-density map from a macromolecular crystal the phase-problem needs to be solved, which often involves the use of heavy-atom derivative crystals and concomitantly the determination of the heavy atom substructure. This is customarily done by direct methods or Patterson-based approaches, which however may fail when only poorly diffracting derivative crystals are available, as often the case for e.g. membrane proteins. Here we present an approach for heavy atom site identification based on a Molecular Replacement Parameter Matrix (MRPM) search. It involves an n-dimensional search to test a wide spectrum of molecular replacement parameters, such as clusters of different conformations. The result is scored by the ability to identify heavy-atom positions, from anomalous difference Fourier maps, that allow meaningful phases to be determined. The strategy was successfully applied in the determination of a membrane protein structure, the CopA Cu+-ATPase, when other methods had failed to resolve the heavy atom substructure. MRPM is particularly suited for proteins undergoing large conformational changes where multiple search models should be generated, and it enables the identification of weak but correct molecular replacement solutions with maximum contrast to prime experimental phasing efforts.Comment: 19 pages total, main paper: 6 pages (2 figures), supplementary material: 13 pages (2 figures, 9 tabels

Glucose transporters: production, crystallization and inhibition

Glucose transporters (GLUTs) comprise a family of 14 membrane proteins that regulate glucose uptake into the cell. Different types of GLUTs are expressed in various tissues and play a crucial role in glucose metabolism. Cancer cells are highly dependant on glucose and therefore GLUTs are possible drug targets for cancer therapy. In order to block the glucose uptake facilitated by GLUTs, various inhibitors are studied and both natural and synthetic compounds having an inhibitory effect on glucose uptake have been discovered. High resolution X-ray structure of the GLUT-inhibitor complex would provide a detailed understanding of protein-inhibitor interactions and contribute to facilitating the development of new derivatives. The focus of this study is on a glucose transporter 1 (GLUT1). The GLUT1 has been produced and crystallization trials set up, which resulted in microcrystals. A series of salicylketoxime based compounds have been shown to inhibit GLUT1 and two lead compounds displaying the highest inhibition have been identified in a giant vesicle assay. The main goal of the study is to determine the structure of the GLUT1 with selected inhibitors. Moreover, studies on one more glucose transporter GLUT3 are carried out to investigate the selectivity of the salcylketoxime compounds

Molecular mechanism of sugar transport in plants unveiled by structures of glucose/H+^+ symporter STP10

Sugars are essential sources of energy and carbon and also function as key signalling molecules in plants. Sugar transport proteins (STP) are proton-coupled symporters responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a vital role in plant responses to stressors such as dehydration and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward-open conformation at 2.6 Å resolution and a structure of the outward-occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with molecular dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. These results advance our understanding of monosaccharide uptake, which is essential for plant organ development, and set the stage for bioengineering strategies in crops