Long-wavelength macromolecular crystallography - First successful native SAD experiment close to the sulfur edge

Abstract

Phasing of novel macromolecular crystal structures has been challenging since the start of structural biology. Making use of anomalous diffraction of natively present elements, such as sulfur and phosphorus, for phasing has been possible for some systems, but hindered by the necessity to access longer X-ray wavelengths in order to make most use of the anomalous scattering contributions of these elements. Presented here are the results from a first successful experimental phasing study of a macromolecular crystal structure at a wavelength close to the sulfur K edge. This has been made possible by the in-vacuum setup and the long-wavelength optimised experimental setup at the I23 beamline at Diamond Light Source. In these early commissioning experiments only standard data collection and processing procedures have been applied, in particular no dedicated absorption correction has been used. Nevertheless the success of the experiment demonstrates that the capability to extract phase information can be even further improved once data collection protocols and data processing have been optimised.

Keywords: Crystallographic phase problem; Macromolecular crystallography; Soft X-rays; Sulfur SAD; X-ray diffraction.

Fig. 1

The anomalous signal, as reported by XSCALE, plotted as a function of resolution for the two datasets. Due to the λ = 4.96 Å dataset covering a larger rotation range than the λ = 1.38 Å dataset, data for both 90° and for 400° are shown. The lower anomalous signal for the 90° section of the λ = 4.96 Å dataset at higher resolution is an effect of the lowered completeness, due to the detector geometry.

Fig. 2

10,000 substructure solution attempts, searching for 9 sites, with SHELXD plotted with CC_weak vs CC_all for the λ = 4.96 Å dataset.

Fig. 3

Experimentally phased map after density modification from SHARP in blue at 1.0 σ and an anomalous difference map at 5.0 σ. The refined thaumatin model is superposed (represented as sticks) to show the protein chain and the position of the sulfur atoms (yellow spheres) in the model. The SHARP map was carved 2.6 Å around the shown residues and the anomalous difference map was carved 3.0 Å around the sulfurs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Experimentally phased anomalous difference map contoured at 5.0 σ shown with the refined model (cartoon representation, coloured by secondary structure) and the positions of the sulfur atoms as yellow spheres. The map was carved with a 2.5 Å cut-off around the sulfur positions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)