Structures of Biological Macromolecules from Multi-crystal Native SAD Phasing

Native metalloproteins have been routine subjects for mutli- and single-wavelength anomalous diffraction (respectively MAD and SAD) analyses for some time; however, despite notable early successes, native molecules without heavy atoms (Z ≥ 25) have only recently become routinely accessible. Crystals of native proteins and nucleic acids have substantial contents of light elements (P, S, Cl, K, Ca) of potential for use in SAD phasing. Anomalous signals from such elements can be enhanced by using a lower than usual x-ray energy; nevertheless, typical Bijvoet differences usually still remain at a level comparable to noise. We have devised robust SAD procedures to study native, light-atom-only biological structures. We have so far used a modestly low energy (6 - 7 keV), but we further enhance the signal-to-noise in anomalous diffraction by combining data from multiple crystals chosen to be statistically equivalent. We have applied our multi-crystal native SAD approach in several structure determinations (1,2) at sizes up to 1200 ordered residues per asymmetric unit and at resolutions so far as low as 3.2 Å. Our tested practices can be replicated readily, and we plan further improvements in computing protocols and in instrumentation.

Native-SAD Phasing at Synchrotrons: A Low-dose and High-redundancy Strategy

Native biological macromolecules contain intrinsic light elements such as sulfur in proteins and phosphorus in nucleic acids. Native-SAD phasing utilizes the anomalous signals from light elements for de novo structure determination: first the substructure of anomalous scatterers is determined; phase evaluation for the entire structure then follows. Synchrotron beamlines are expected to be ideal instruments for native-SAD phasing due to their brilliant and energy-tunable x-rays. However, anomalous signals from light elements are typically very weak at x-ray energies accessible to most synchrotron beamlines. Efforts have been made to promote the utility of synchrotrons for routine native-SAD phasing with no requirement for heavy-atom incorporation. Our strategy is to limit the x-ray dose per crystal and to enhance the signal-to-noise ratio by increasing data redundancy through use of multiple crystals. We have devised a robust procedure and applied it for routine native-SAD analyses on real-life membrane proteins, protein-protein complexes, and recalcitrant proteins. Here we use these real-life case studies to illustrate our procedures in sample preparation, x-ray energy selection, data collection, data analysis and phasing.