Abstract
Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography. Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was used mainly to supplement the isomorphous replacement or to locate the anomalous scatterer itself. The first step in solving macromolecular structures by SAD (single-wavelength anomalous diffraction) is the location of the anomalous scatterers. The SAD method for experimental phasing has evolved substantially in the recent years. A phasing tool, 5-amino-2,4,6- triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with proteins. The high quality of the diffraction data, the use of chromium anode X-ray radiation and the required amount of anomalous signal enabled way for successful structure determination and automated model building. An analysis and/or comparison of the sulfur and iodine anomalous signals at the Cr Kα wavelength are discussed.
Highlights
Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible
A phasing tool, 5-amino-2,4,6triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with proteins
Glucose Isomerase and Thermolysin crystals appeared after a day and belonged to the tetragonal P43212, orthorhombic I222 and hexagonal P6122 space groups, respectively with one molecule per asymmetric unit
Summary
Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. It is not easy to obtain automatically the crystal derivatives, which is appropriate for phasing. New ideas have been put forward, that aim in making the phasing of novel structures easier and more susceptible to routine and automatic treatment [1]. Phase problem is a bottleneck in macromolecular structure determination and in model building which is a time-consuming task. Phases can be derived from some knowledge of the molecular structure. Structures of small proteins (molecular weight less than 10 kDa) can be determined in solution using nuclear magnetic resonance (NMR) spectroscopy and the assembly of proteins in a complex can be studied using electron microscopy, but only X-ray diffraction helps in determining the three dimensional structure of small and large proteins with a precision of about 0.1 - 0.2 Å. The phases are derived either by Molecular
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