Experimental phasing with SHELXC/D/E: combining chain tracing with density modification

George M Sheldrick

doi:10.1107/s0907444909038360

Abstract

The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue alpha-helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a 'no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.

Highlights

Experimental phasing of macromolecules usually requires the presence of marker atoms such as metal atoms or sulfur in a native protein, heavy metals or halides introduced by soaking or selenium incorporated by replacing methionine with selenomethionine using a suitable expression system
The programs are restricted to experimental phasing by MAD, SAD, SIR, SIRAS and RIP methods
The program SHELXC provides a statistical analysis of the input data, estimates the marker-atom structure factors FA and the phase shifts and doi:10.1107/S0907444909038360 479 research papers sets up the files for the other two programs

Summary

Introduction

Experimental phasing of macromolecules usually requires the presence of marker atoms such as metal atoms or sulfur in a native protein, heavy metals or halides introduced by soaking or selenium incorporated by replacing methionine with selenomethionine using a suitable expression system. In the program suite SHELXC/D/E (Sheldrick, 2008), every attempt has been made to reduce experimental phasing to its absolute essentials, with the aim of obtaining an interpretable electron-density map quickly and reliably rather than finding the most accurate phases This requires some severe simplifications, for example the assumption that only one type of marker atom is present, in practice a mixture of elements rarely causes problems. An electron-density map calculated using these approximate phases ’T and the observed structure factors FT may well be difficult or impossible to interpret This is especially true for SAD phasing, where the estimates of are restricted to 90 (when reflection h, k, l is significantly stronger than reflection Àh, Àk, Àl) or 270 (when the opposite is true); these estimates are more reliable when the anomalous difference is large. In this paper an alternative approach, the sphere-of-influence method (Sheldrick, 2002), will be extended by iterating it with main-chain tracing

Experimental phase information

Substructure solution

The sphere-of-influence algorithm

Findings

Autotracing

Conclusions