Structure Determination of a 16.8 kDa Copper Protein at 2.1 Å Resolution Using Anomalous Scattering Data with Direct Methods

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 Å resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper ( = 3.9 e⁻) and then conventionally refined (R_cryst = 18.7%, R_free = 21.9%). This is the largest unknown protein structure (M_r ≃ 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (∼2 Å) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper ( ≃ 4 e⁻), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se ( = 8 e⁻ at the `white line') and 80 kDa for a Pt derivative ( = 19 e<sup>−</sup> at the `white line', L₃ edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.