Making routine native SAD a reality: lessons from beamline X06DA at the Swiss Light Source.

Shibom Basu,Laura Vera,Vincent Olieric,Meitian Wang,Aaron Finke

doi:10.1107/s2059798319003103

Abstract

Native single-wavelength anomalous dispersion (SAD) is the most attractive de novo phasing method in macromolecular crystallography, as it directly utilizes intrinsic anomalous scattering from native crystals. However, the success of such an experiment depends on accurate measurements of the reflection intensities and therefore on careful data-collection protocols. Here, the low-dose, multiple-orientation data-collection protocol for native SAD phasing developed at beamline X06DA (PXIII) at the Swiss Light Source is reviewed, and its usage over the last four years on conventional crystals (>50 µm) is reported. Being experimentally very simple and fast, this method has gained popularity and has delivered 45 de novo structures to date (13 of which have been published). Native SAD is currently the primary choice for experimental phasing among X06DA users. The method can address challenging cases: here, native SAD phasing performed on a streptavidin-biotin crystal with P21 symmetry and a low Bijvoet ratio of 0.6% is highlighted. The use of intrinsic anomalous signals as sequence markers for model building and the assignment of ions is also briefly described.

Highlights

Single-wavelength anomalous dispersion (SAD) is the most popular experimental phasing technique for de novo structure determination by macromolecular crystallography (MX)
To overcome these limitations and push the limit of native single-wavelength anomalous dispersion (SAD) phasing (Bent et al, 2016), special setups designed for native SAD data collection at energies below 5 keV have been developed at beamlines I23 at Diamond Light Source in the UK
We and our users have routinely been using our native SAD strategy for the last four years, and we have already reported some of our major successes (Weinert et al, 2015; Olieric et al, 2016)

Summary

Introduction

Single-wavelength anomalous dispersion (SAD) is the most popular experimental phasing technique for de novo structure determination by macromolecular crystallography (MX). The vast majority of these SAD experiments exploited the strong anomalous signal of heavy atoms, delivered either by soaking of heavy elements or by the expression of selenomethionyl protein (Hendrickson, 2014) These derivatizations can be problematic owing to loss of both isomorphism and diffraction. Other difficulties in low-energy crystallography are on the detector side, with both inaccurate calibration and a loss of high-angle diffraction with a typical flat configuration, owing to the increasing Bragg angles of reflection To overcome these limitations and push the limit of native SAD phasing (Bent et al, 2016), special setups designed for native SAD data collection at energies below 5 keV have been developed at beamlines I23 at Diamond Light Source in the UK

Methods

Results

Conclusion