Automated refinement of macromolecular structures at low resolution using prior information.

Oleg Kovalevskiy,Garib N Murshudov,Robert A Nicholls

doi:10.1107/s2059798316014534

Abstract

Since the ratio of the number of observations to adjustable parameters is small at low resolution, it is necessary to use complementary information for the analysis of such data. ProSMART is a program that can generate restraints for macromolecules using homologous structures, as well as generic restraints for the stabilization of secondary structures. These restraints are used by REFMAC5 to stabilize the refinement of an atomic model. However, the optimal refinement protocol varies from case to case, and it is not always obvious how to select appropriate homologous structure(s), or other sources of prior information, for restraint generation. After running extensive tests on a large data set of low-resolution models, the best-performing refinement protocols and strategies for the selection of homologous structures have been identified. These strategies and protocols have been implemented in the Low-Resolution Structure Refinement (LORESTR) pipeline. The pipeline performs auto-detection of twinning and selects the optimal scaling method and solvent parameters. LORESTR can either use user-supplied homologous structures, or run an automated BLAST search and download homologues from the PDB. The pipeline executes multiple model-refinement instances using different parameters in order to find the best protocol. Tests show that the automated pipeline improves R factors, geometry and Ramachandran statistics for 94% of the low-resolution cases from the PDB included in the test set.

Highlights

Poor diffraction quality from macromolecular crystals is a persistent problem: various types of short-range and longrange disorder induced by impurities, imperfect crystal-growth conditions and the natural conformational mobility of macromolecules result in the weakening of high-resolution observations, anisotropic diffraction and other problems (Chernov, 2003; Shaikevitch & Kam, 1981; Caylor et al, 1999)
The best-performing protocol that showed the maximal improvement in Rfree for the maximal number (37.4%) of structures in the test set was protocol 3: refinement with external restraints from a single homologue followed by a second round of refinement using jelly-body restraints
We found that in cases where high-resolution homologues are available the best strategy is to first execute a REFMAC5 refinement run using external restraints generated by ProSMART, followed by a second round of refinement using only jelly-body restraints

Summary

Introduction

Poor diffraction quality from macromolecular crystals is a persistent problem: various types of short-range and longrange disorder induced by impurities, imperfect crystal-growth conditions and the natural conformational mobility of macromolecules result in the weakening of high-resolution observations, anisotropic diffraction and other problems (Chernov, 2003; Shaikevitch & Kam, 1981; Caylor et al, 1999). Poor crystal diffraction results in the corresponding data sets having a low information content. Whilst substantial effort has been directed towards improving crystal diffraction quality (Heras & Martin, 2005), quite often it is technically impossible to achieve high-quality diffraction, especially for large multisubunit complexes. The primary aim of X-ray structure analysis is not to obtain the perfect crystal, but rather to determine atomic models of proteins, nucleic acids or complexes of interest that are of sufficient quality to be of use in addressing questions of biological relevance. There is a high demand for the development of techniques to allow crystallographers to build reliable models using incomplete, limited and noisy diffraction data

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Conclusion