STRUCTURE DETERMINATION OF A 16.8 KDA COPPER PROTEIN AT 2.1 ANGSTROM 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 equa l to 16.8 kDa) to be determined using the SAS and direct-methods appro ach and demonstrates that by exploiting the anomalous signal at a sing le wavelength, direct methods can be used to determine phases at typic al (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 c ould be used for proteins with molecular weights of up to 33 kDa per S e (f ''(max) = 8 e(-) at the 'white line') and 80 kDa for a Pt derivat ive (f ''(max) = 19 e(-) at the 'white line', L-3 edge). The method pr ovides a powerful alternative in solving a de novo protein structure w ithout either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anoma lous diffraction (MAD) data.