Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing.

Thomas C. Terwilliger,David L. Akey,Gábor Bunkóczi,Peter H. Zwart,Li-Wei Hung,Janet L. Smith,Paul D. Adams

doi:10.1107/s2059798315019269

Abstract

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.

Highlights

The useful anomalous correlation calculate the useful anomalous correlation (CCano) is the correlation between measured anomalous differences and those expected of an ideal structure where the only anomalous scatterers are those in the anomalous substructure and where there are no errors in measurement (29)
The number of reflections Nrefl includes all acentric reflections that are unique under space-group symmetry, and the number of sites nsites is the number contained in the asymmetric unit of the crystal
356 Terwilliger et al Anomalous signal in single-wavelength anomalous diffraction (SAD) phasing differences with those corresponding only to the anomalous substructure, the number of unique reflections measured, the number of sites in the substructure and the atomic displacement factors for the atoms in the substructure. Combining this with the empirical observation that the anomalous signal is a predictor of solving the anomalous substructure (Fig. 2) gives a clear idea of the features of the crystal and the experiment that determine whether the substructure can be obtained. (31) shows that even if the data are measured precisely, the anomalous signal is limited by several factors

Summary

Introduction

The single-wavelength anomalous diffraction (SAD) method is a remarkably powerful approach to macromolecular structure determination (Hendrickson & Teeter, 1981; Wang, 1985; reviewed in Dauter et al, 2002; Hendrickson, 2014) It has become the dominant method for the determination by X-ray diffraction of structures that are not closely related to any structure already present in the Protein Data Bank (PDB; Berman et al, 2000), accounting for 73% of such new structures by 2013. Depending on the element and the wavelength, part of the scattering from the atoms in this substructure will be shifted in phase by /2 relative to the ‘normal’ scattering from other atoms in the structure This results in a difference in intensities for reflections related by inversion which otherwise would have identical intensities (Bijvoet, 1954).

Objectives

Methods

Results

Conclusion