Protein structure of NK1 fragment of HGF/SF (Hepatocyte Growth Factor/Scatter Factor, PDB-ID codes: 1nk1, 1bht) has been successfully determined using Sulphur SAD (S-SAD) experiments on the in-house Cu Kalpha source (i.e. ICARUS). The protein has molecular weight of about 25 kDa and contains 12 sulphur atoms (Met and Cys). The crystals belong to P21 spacegroup and the asymmetric contains a dimer of NK1.
The S-SAD data collection was performed on ICARUS (X8 PROTEUM X-ray diffraction system). The NK1 crystal diffracted to about 2.0 Angstrom resolution (<I/sigma> in the high resolution shell is 3.5). A total of 6,079 diffraction images were recorded which took 2 days and 12 hours to acquire. The dataset’s overall redundancy is 46.
The search for the sulphur atoms as well as phasing and electron density modifications were performed using PHENIX (v1.7-650). The anomalous signal was of reasonable strength only up to 3.5 Angstrom resolution. The positions of 13 sulphur atoms (out of 24 possible) were identified, resulting in a set of phases with the figure of merit (FOM) of 0.41.
Here are the density modified S-SAD maps:
The autobuilding procedure by PHENIX using all data to 2.0 Angstrom resolution built 90% of the structure resulting in Rcryst of 23 % and Rfree of 28%.
Here is a sample of final 2Fo-Fc electron density maps:
It is indeed possible to solve protein crystal structure via Sulphur SAD using Cu Kalpha source. However, as the anomalous signal of sulphur is extremely small (delta f’’ is only 0.5 e- at Cu Kalpha), great care should be taken during the data collection, i.e. the overall dataset redundancy should be more than 30 and the crystal must survive for the duration of the data collection experiment.