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

1998; Wiley; Volume: 54; Issue: 4 Linguagem: Inglês

10.1107/s0907444998005423

1399-0047

Ian Harvey, Quan Hao, Elizabeth Duke, W. John Ingledew, S.S. Hasnain,

Cultural Heritage Materials Analysis

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^{\prime\prime} = 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^{\prime\prime} ≃ 4 e−), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f_{\rm max}^{\prime\prime} = 8 e− at the `white line') and 80 kDa for a Pt derivative (f_{\rm max}^{\prime\prime} = 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.