Abstract
Increasing sophistication in molecular-replacement (MR) software and the rapid expansion of the PDB in recent years have allowed the technique to become the dominant method for determining the phases of a target structure in macromolecular X-ray crystallography. In addition, improvements in bioinformatic techniques for finding suitable homologous structures for use as MR search models, combined with developments in refinement and model-building techniques, have pushed the applicability of MR to lower sequence identities and made weak MR solutions more amenable to refinement and improvement. MrBUMP is a CCP4 pipeline which automates all stages of the MR procedure. Its scope covers everything from the sourcing and preparation of suitable search models right through to rebuilding of the positioned search model. Recent improvements to the pipeline include the adoption of more sensitive bioinformatic tools for sourcing search models, enhanced model-preparation techniques including better ensembling of homologues, and the use of phase improvement and model building on the resulting solution. The pipeline has also been deployed as an online service through CCP4 online, which allows its users to exploit large bioinformatic databases and coarse-grained parallelism to speed up the determination of a possible solution. Finally, the molecular-graphics application CCP4mg has been combined with MrBUMP to provide an interactive visual aid to the user during the process of selecting and manipulating search models for use in MR. Here, these developments in MrBUMP are described with a case study to explore how some of the enhancements to the pipeline and to CCP4mg can help to solve a difficult case.
Highlights
Molecular replacement (MR) is one of the key methods used to solve the phase problem in macromolecular crystallography (MX)
The only program options set by MrBUMP are the anticipated number of molecules expected in the asymmetric unit, which is calculated using MATTHEWS_COEF (Matthews, 1968; Kantardjieff & Rupp, 2003), and the expected r.m.s.d. value for each search model given to Phaser
To evaluate how best to use the capabilities of this method of running MrBUMP, we explored its use for the case of PDB
Summary
Molecular replacement (MR) is one of the key methods used to solve the phase problem in macromolecular crystallography (MX). (Jenkins, 2018) uses fragment libraries, rapidly processing them in Phaser and attempting to improve the resulting phases with ACORN (Foadi et al, 2000; Yao et al, 2005) Another developing trend in unconventional methods is the brute-force trialling of a library of all (or a nonredundant subset) of the set of known structures in MR against a target. It has been known for a while that MR search models clustered and aligned into ensembles can be more effective than the individual models themselves (Leahy et al, 1992; Pieper et al, 1998; Chen et al, 2000; Rigden et al, 2002; Bibby et al, 2012; Keegan et al, 2015) Phaser can exploit this alignment to produce a statistically weighted structure-factor set based on the variance across the aligned models (Read, 2001). In cases where the user’s estimates are too low to be consistent, more appropriate values are derived from the computed structure factors of the ensemble
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