Recent developments in MrBUMP: better search-model preparation, graphical interaction with search models, and solution improvement and assessment

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.

Keywords: CCP4 pipeline; MrBUMP; automated structure solution; molecular replacement.

Figure 1

A simple flowchart representation of the MrBUMP pipeline. The program can be run in two modes: model search and preparation only or model search, preparation, MR, refinement and model building.

Figure 2

The CCP4mg interface to MrBUMP, with an illustrative representation of the domain regions found during the clustering of matches from the phmmer search. The results shown here are for a search using the sequence from PDB entry 5u4p, a protein–protein complex between 26S proteasome regulatory subunit RPN8, RPN11 and ubiquitin S31 (Worden et al., 2017 ▸). The results have been clustered into four domains, two of which consist of more than one match, making them suitable for ensemble generation. The dashed line indicates the cutoff phmmer score (default = 20) used for the selection of matches to be used as search models in MR.

Figure 3

The CCP4mg/MrBUMP interface displaying the results of a search using the sequence from PDB entry 5cml. Search models found in the search step are pruned using Sculptor and aligned using the GESAMT structure-alignment program. The resulting ensemble consists of ten individual search models derived from the following chains: chains A and B of PDB entries 3pf8, 3pfb and 3s2z, chain A of PDB entries 3pfc, 3qm1 and 3pf9, and chain B of PDB entry 2wtn.

Figure 4

Three levels of the truncated ensemble model (ice blue) aligned with chain A of PDB entry 5cml (orange/yellow). (a) Base ensemble, (b) 80% truncation level, (c) 40% truncation level.

Figure 5

Colour maps of various scores and values of the input Phaser r.m.s.d. against the truncation level for ensemble and single models. Results are displayed for the final variance-r.m.s. (VRMS) value from Phaser (a, b), the final log-likelihood gain (LLG) scores from Phaser (c, d), the final R _free value from REFMAC after 100 cycles using jelly-body restraints (e, f), the SHELXE CC between the native structure factors and those calculated from the output polyalanine trace model (g, h) and the SHELXE output polyalanine trace model average chain length (ACL) (i, j). As a reference, the white boxes show the r.m.s.d. estimate calculated by pairwise alignment in GESAMT for the single search model against the target structure at each truncation level.

Figure 6

The map correlation coefficient (mapCC) for the electron-density maps generated after the Phaser (a, b), REFMAC (c, d) and SHELXE (e, f) steps compared with a map calculated from the deposited intensities and structure for PDB entry 5cml. Results are shown for all input Phaser r.m.s.d. values and all truncation levels (ensemble and single search models). The colour plots illustrate how the map CC increases after each step for most of the correctly placed MR solutions. The map coefficients generated by both Phaser and REFMAC were used in the comparison. The deposited amplitudes were used in combination with the calculated phases from SHELXE to generate a map for the SHELXE comparison. As a reference, the grey boxes show the r.m.s.d. estimate calculated by pairwise alignment in GESAMT for the single search model against the target at each truncation level.