In-house sulfur SAD phasing: a case study of the effects of data quality and resolution cutoffs

Abstract

Single-wavelength anomalous diffraction (SAD) utilizing the weak signal of inherently present S atoms can be successfully used to solve macromolecular structures, although this is mostly performed with data from a synchrotron rather than a laboratory source. Using high redundancy, sufficiently accurate anomalous data may now often be collected in the laboratory using Cu Kalpha X-ray radiation. Systematic analyses of a laboratory-derived data set illuminate the effects of data quality, redundancy and resolution cutoffs on the ability to locate the S atoms and phase the structure of Ptr ToxA, a 13.2 kDa toxin secreted by the fungus Pyrenophora tritici-repentis. Three sulfurs contributed to the successful phasing of the structure and were located using the program SHELXD. It is observed that data quality improves with increasing redundancy, but after a certain point becomes worse owing to crystal decay, so that there is an optimal amount of data to include for the sulfur substructure solution. Further, the success rate in locating S atoms is dramatically improved at lower resolutions and in a manner similar to data quality, there exists an optimal resolution at which the likelihood of solving the substructure is maximized. Based on these observations, a strategy for SAD data collection and substructure solution is suggested.

A multi-dataset data-collection strategy produces better diffraction data

“…(iii) Data-collection strategy: the wavelength, attenuation, detector-to-crystal distance, exposure time, start angle, scan range and oscillation angle. Therefore, for a given crystal and X-ray data-collection system, the key to obtaining the highest possible quality of diffraction data lies in the datacollection strategy (Cianci et al, 2008;Sarma & Karplus, 2006).…”

An overview of heavy-atom derivatization of protein crystals

“…Recently, it has also become possible to solve structures using the anomalous signal from the native S atoms in methionines and cysteines in a protein (Hendrickson & Teeter, 1981;Debreczeni et al, 2003;Sarma & Karplus, 2006;Goulet et al, 2010), although sulfur would not normally be regarded as a HA. The development of long-wavelength beamlines at synchrotrons (such as I23 at the Diamond Light Source; Wagner et al, 2016) specifically designed for sulfur phasing will further increase the popularity of this method.…”

In-House Zinc SAD Phasing at Cu Kα Edge

“…SAD phasing has been reported at remote wavelengths above the absorption edge (Leonard et al, 2005) and even at Cu Kα edge of in-house X-ray sources (Dauter et al, 2002;Debreczeni et al, 2003a;2003c;Evans and Bricogne, 2003;Roeser et al, 2005;Sarma and Karplus, 2006;Stevenson et al, 2004;Vennila and Velmurugan, 2011;Yogavel et al, 2007;2010a;2010b). The successful in-house SAD phasing opens new avenue of experimental phasing with in-house X-ray sources.…”