SMMS, University of Queensland, Australia* IMB, University of Queensland, Australia** SBS, Flinders University of South Australia, Australia*** Division of Plant Industry, CSIRO, Australia****
○Gregor Guncar*,** Ching-I A. Wang* Jade K. Forwood*,** Trazel Teh* Ann-Maree Catanzariti**** Horst Schirra** Peter A. Anderson*** Jeffrey G. Ellis**** Peter N. Dodds**** and Bostjan Kobe*,**
X-ray anomalous scattering from a single cobalt ion was used for phasing of the Melampsora lini (flax rust) avirulence protein Avr567-A. AvrL567-A is a protein from a fungal rust pathogen that induces gene-for-gene based mechanism of plant disease resistance in flax, where it is recognised by plant resistance proteins (L5, L6 and L7). In crystallization trials we observed that AvrL567-A is a cobalt binding protein. Cobalt absorption edge is close to CuKα wavelength, therefore we were able to collect a 2.0 Å resolution single-wavelength anomalous dispersion dataset using a conventional in- house copper rotating anode X- ray generator. Phases calculated using the program Solve were sufficient for Resolve to automatically build most of the structure. Native datasets at 1.7 Å and 1.4 Å resolution were used subsequently to build the rest of the model using program Arp/Warp. The Avr567-A structure reveals a new β-sandwich-like protein fold and sheds light on the structural basis of recognition specificity of the L proteins for the AvrL567 family members. It also provides insights into possible pathogenassociated functions of the AvrL567 proteins. We demonstrate that Co2+ can be used for SAD phasing using Cu rotating anode, which is advantageous when synchrotron radiation may not be readily available the method is applicable to proteins binding metals that can be substituted with cobalt.