Applications of anomalous scattering from S atoms for improved phasing of protein diffraction data collected at Cu K α wavelength

The anomalous signal of S atoms is easily detected at the Cu  K α wavelength of a non‐synchrotron source with current data‐collection methods. The position of sulfur and other anomalous scatterers can be located through an anomalous difference Fourier map ( F + − F − , ϕ calc − 90°). It has been discovered experimentally that even low‐quality preliminary phases are often sufficient to find anomalous scatterers. Their anomalous signal in the native crystal can contribute to significant improvement in phase refinement. This technique has been applied to solve the crystal structures of orthorhombic lysozyme and thaumatin. Furthermore, the structure of trypsin was solved using only the diffraction data set from a native crystal collected at a single wavelength (Cu  K α) from a rotating‐anode X‐ray generator. The anomalous scattering of sulfur was essential to solve the structure of trypsin which was initially phased from a single intrinsic Ca 2+ atom. The positions of the S atoms of lysozyme and thaumatin were found using the initial SIRAS phases and used in phase refinement. The overall figures of merit and those in each resolution shell were consistently improved. This resulted in much improved electron‐density maps even when the diffraction data were limited to 2.5 Å resolution or worse. Furthermore, peaks from S atoms and other anomalous scatterers in anomalous difference Fourier maps can confirm the tracing of the peptide chain and also provide independent unbiased confirmation of molecular‐replacement results. Thus, the anomalous signal of S atoms can contribute to many aspects of solving protein structures and should be used routinely.