The tertiary structure of azurin from Pseudomonas denitrificans as determined by Cu resonant diffraction using synchrotron radiation

Abstract

The structure of Pseudomonas denitrificans azurin has been solved at 3.0 Å resolution by film data collection methods using synchrotron radiation. Bijvoet pairs of reflections were collected using 8.98 keV radiation where both Δ f′ and Δ f″, the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller. The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections. An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 Å resolution were subsequently calculated from the initial model. The atomic model was subsequently rebuilt using all observed data. The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement.