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 Angstrom resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f " = 3.9 e(-)) and then conventionally refined (R-cryst = 18.7%, R-free = 21.9%). This is the largest unknown protein structure (M-r similar or equal to 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 (similar to 2 Angstrom) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f " similar or equal to 4 e(-)), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f "(max) = 8 e(-) at the 'white line') and 80 kDa for a Pt derivative (f "(max) = 19 e(-) at the 'white line', L-3 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.