Structure determination of a 16.8 kDa copper protein at 2.1 a resolution using anomalous scattering data with direct methods

Abstract

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 (f″ = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr ≃ 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 (f″ ≃ 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', 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. © 1998 International Union of Crystallography all rights reserved.