An introduction to experimental phasing of macromolecules illustrated bySHELX; new autotracing features

Abstract

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. TheSHELXprograms provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e.those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

Evaluation of template‐based modeling in CASP13

“…In MR, an atomic model derived from a related protein structure is rotated and translated in a search for the position occupied by the true structure in the crystal; phases calculated from the model are combined with data to produce an electron density map that reveals new features, if the model is sufficiently accurate. MR, when it succeeds, allows a structure to be determined from a data set from a single native crystal, without requiring the preparation of heavy‐atom derivatives or the accurate measurement of anomalous scattering data . TBM methods that improve significantly on the original template can therefore shortcut the process of structure determination and improve throughput in X‐ray crystallography.…”

“…MR, when it succeeds, allows a structure to be determined from a data set from a single native crystal, without requiring the preparation of heavy-atom derivatives or the accurate measurement of anomalous scattering data. 12 TBM methods that improve significantly on the original template can therefore shortcut the process of structure determination and improve throughput in X-ray crystallography. In recognition of this, in CASP7 we introduced a metric scoring individual model predictions based on their usefulness in MR. 6 As a result of continuing improvement in structure prediction, the use of TBM to improve MR models has been greatly expanding in recent years.…”

Fe-S protein assembly involves bipartite client binding and conformational flexibility in the CIA targeting complex

“…The structure was solved by single-wavelength anomalous dispersion (SAD) phasing using a dataset obtained from Ta 6 Br 12 -derivatized crystals of the Mm MMS19- Mm CIAO1-CIAO2B complex. Heavy atom search was performed in SHELXD and phasing was carried out in SHARP 51, 52 . The resulting phases were improved by density modification with Parrot as implemented in ccp4 53 .…”

Identification of the active site residues in ATP‐citrate lyase's carboxy‐terminal portion

“…Additional degrees of freedom modified the fragments during molecular replacement through gyre refinement against the rotation function and gimble refinement after placement . Best scored phase sets were subject to density modification and autotracing with SHELXE leading to a main chain trace comprising 672 residues and characterized by a CC of 31%. The model and electron density maps were visualized using COOT and the model was refined using phenix.refine .…”