Soft X-rays, high redundancy, and ways & means proper scaling: A new procedure for automated protein structure determination via SAS

A method is described that in our opinion has the potential to become a valuable addition to the nowadays still small arsenal of procedures that can be employed to determine a novel intermediate-sized protein structure completely automatically. The three main features of the method are: (i) the use of soft X-rays in the wavelength range λ=1.5-3.0 Å for diffraction data collection, (ii) the collection of highly redundant diffraction data, and (iii) a suitable scaling protocol for performing a pseudo-absorption correction. This approach was tested on hexagonal crystals of the Zn-metalloprotease Thermolysin. The complete anomalous substructure (one Zn²⁺, five Ca²⁺, three S) could be determined based on anomalous intensity differences as small as 1.3%. All combinations of data collection wavelength and maximum resolution tried were successful. Phase determination was then carried out with standard programs, and the model was built automatically. The only manual intervention necessary was the shuffling of the data between the different computer programs. No evaluation of the intermediate results was necessary at any stage. After the autobuilding stage, the model was nearly complete, and building of the remaining parts appeared to be straightforward. The time required for completing and checking the model on a graphics terminal was estimated to be less than an hour or two. The described approach requires only modestly high-resolution data; it should therefore be applicable to a wide range of protein crystals that contain up to 35 kDa (or maybe even more) in their asymmetric unit.