A practical phasing procedure using the MAD method without the aid of XAFS measurements: successful solution in the structure determination of the outer-membrane lipoprotein carrier LolA.

Abstract

A practical procedure for MAD phasing was successfully performed in the structure determination of the LolA protein, even though the XAFS data could not be measured owing to the overlap of the fluorescence spectra of the atoms that contribute significant signals for anomalous dispersion. The LolA protein, a periplasmic chaperone functioning as an outer-membrane lipoprotein carrier of the Lol system which mediates translocation of the water-insoluble outer-membrane lipoprotein across the periplasm in Gram-negative bacteria, was crystallized in two forms: orthorhombic (I222) and trigonal (P3(1)21 or P3(2)21). A multi-wavelength data set was collected from a platinum derivative of the orthorhombic crystals grown from a buffer solution containing zinc acetate and cacodylate (an arsenic compound), but XAFS measurements could not be performed because the energies of the fluorescence spectra of Zn atoms, As atoms and Pt atoms are in very close proximity. However, effective MAD data were collected with six wavelength data sets near the platinum absorption edge and the f' and f" values could subsequently be estimated from the data statistics and the peak height of the dispersive and anomalous difference Patterson maps. The subsequent MAD phasing gave a high-quality initial electron-density map which was sufficient to construct a complete molecular model of the LolA protein.