Structure determination of a 16.8 kDa copper protein at 2.1 A resolution using anomalous scattering data with direct methods.
The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 A 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 approximately /= 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 (approximately 2 A) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f" approximately 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.
School of Applied Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, England, and CLRC Daresbury Laboratory, Warrington WA4 4AD, England.
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I
Harvey
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Hao
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Duke
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Ingledew
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Hasnain
D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011487:Protein Conformation; D013855:Thiobacillus / Q000737:chemistry
D001426:Bacterial Proteins; C012543:rusticyanin; D001400:Azurin