Anomalous Dispersion Experiment (anomalous + dispersion_experiment)

Distribution by Scientific Domains

Selected Abstracts

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membrane

Muhammad Bashir Khan
Mrs2 transporters are distantly related to the major bacterial Mg2+ transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16, resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal. [source]

Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461

Joana Rocha
Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40, resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein. [source]

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a resuscitation-promoting factor from Mycobacterium tuberculosis

Alessia Ruggiero
The resuscitation-promoting factor RpfB, the most complex of the five resuscitation-promoting factors produced by M. tuberculosis, is devoted to bacterial reactivation from the dormant state. RpfB consists of 362 residues predicted to form five domains. An RpfB fragment containing the protein catalytic domain and a G5 domain has been successfully crystallized using vapour-diffusion methods. This is the first crystallographic study of a resuscitation-promoting factor. Crystals of this protein belong to space group I422, with unit-cell parameters a = 97.63, b = 97.63, c = 114.87,. Diffraction data have also been collected from a selenomethionine derivative at 2.9, resolution. Model building using the phases derived from the multiwavelength anomalous dispersion experiment is in progress. [source]

The interdependence of wavelength, redundancy and dose in sulfur SAD experiments

Michele Cianci
In the last decade, the popularity of sulfur SAD anomalous dispersion experiments has spread rapidly among synchrotron users as a quick and streamlined way of solving the phase problem in macromolecular crystallography. On beamline 10 at SRS (Daresbury Laboratory, UK), a versatile design has allowed test data sets to be collected at six wavelengths between 0.979 and 2.290, in order to evaluate the importance and the interdependence of experimental variables such as the Bijvoet ratio, wavelength, resolution limit, data redundancy and absorbed X-ray dose in the sample per data set. All the samples used in the experiments were high-quality hen egg-white lysozyme crystals. X-radiation damage was found to affect disulfide bridges after the crystals had been given a total dose of 0.20 107,Gy. However, with such a total dose, it was still possible in all cases to find a strategy to collect data sets to determine the sulfur substructure and produce good-quality phases by choosing an optimum combination of wavelength, exposure time and redundancy. A ,|,ano|/,(,ano), greater than 1.5 for all resolution shells was a necessary requirement for successful sulfur SAD substructure location. Provided this is achieved, it seems possible to find an optimum compromise between wavelength, redundancy and dose to provide phasing information. The choice of the wavelength should then follow the sample composition and the diffracting properties of the crystal. For strongly diffracting crystals, wavelengths equal or shorter than 1.540, can be selected to capture the available data (provided the Bijvoet ratio is reasonable), while a longer wavelength, to gain as high a Bijvoet ratio as possible, must be used for more weakly diffracting crystals. These results suggest that an approach to a sulfur SAD experiment based on a complete description of the crystal system and the instrument for data collection is useful. [source]