Abstract
Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine-carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.
Highlights
Experimental phasing is vital for the determination of threedimensional protein structures using single-crystal X-ray diffraction
Data sets were prepared with XPREP (Bruker AXS Inc., Madison, Wisconsin, USA)
B3C and I3C represent a novel class of compounds that show interaction with protein molecules
Summary
Experimental phasing is vital for the determination of threedimensional protein structures using single-crystal X-ray diffraction. With the latest advances in synchrotron hardware, e.g. improved detectors, phasing with the weak anomalous signal from intrinsic scatterers has become a possible option, high-quality data are required. These are more readily obtained when high symmetry enables a high data redundancy to be achieved, but low-symmetry examples have proved successful (Lakomek et al, 2009). Tereshko et al, 2008; Schuermann & Tanner, 2003; Roversi et al, 2010), and is included in the Auto-Rickshaw server (Panjikar et al, 2009) Since most of these are specialized applications and are not generally applicable, a quick and easy approach towards experimental phasing would still be desirable for the derivatization of protein crystals and experimental phase determination
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