The use of Co2+ for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Abstract

Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 A) is compatible with the Cu K alpha wavelength (1.54 A) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.

Figure 1

Representative crystal of Co-AvrL567-A grown in 10% PEG 8000, 0.1 M imidazole pH 8.5 and 17.5 mM CoCl₂. The blue colour suggested tetrahedral Co²⁺ coordination.

Figure 2

The incorporation of cobalt can be detected by MALDI–TOF mass spectrometry. The result shows the cobalt:protein stoichiometry to be 1:1. The error of the measurement is less than 1 Da (the cobalt-bound AvrL567-A has a molecular weight of 14 568.41 Da).

Figure 3

Resolution-dependence of the anomalous signal-to-noise ratio as calculated using the program SHELXC (Schneider & Sheldrick, 2002 ▶).

Figure 4

The tetrahedral cobalt coordination-facilitated crystal contacts. The figure shows a stereoview of the SAD electron density (following density modification with DM and RESOLVE) at 2.0 Å resolution contoured at 1.0σ (black) and the anomalous map contoured at 5.0σ (magenta); superimposed is the refined model of AvrL567-A shown in stick representation. Cobalt is shown as a green sphere and C atoms from the symmetry-related molecule are shown in green, otherwise they are shown in light blue; N, O and S atoms are shown in dark blue, red and yellow, respectively. The imidazole molecule at the top comes from the crystallization solution.