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SAD-phasing of Zn-substituted Pseudoazurin and the Influence of Data Multiplicity

SC-XRD Application Note


Pseudoazurin is a blue-copper protein found in the periplasm of denitrifying bacteria. It participates in the electron transport process leading to the reduction of nitrite to mainly nitric oxide. For the requirements of various spectroscopy experiments, substitution of the original Cu(II) by alternative transition metal ions has been carried out successfully. In order to examine the phasing potential of the very weak anomalous signal of sulphur (Δf" ~0.56 e-) and zinc (Δf" ~0.68 e-) atoms using an in-house Cu-Kα radiation source, we substituted the native Cu2+ of pseudoazurin by Zn2+ ion and determined its crystal structure1 by Zn/S SAD-phasing at 2.2 Å. To achieve this, Single wavelength Anomalous Dispersion (SAD) datasets have been initially collected with high multiplicity. The lowest multiplicity required to obtain the minimal Zn/S substructure for structure determination was also investigated.