The JCSG MR pipeline: optimized alignments, multiple models and parallel searches

Abstract

The success rate of molecular replacement (MR) falls considerably when search models share less than 35% sequence identity with their templates, but can be improved significantly by using fold-recognition methods combined with exhaustive MR searches. Models based on alignments calculated with fold-recognition algorithms are more accurate than models based on conventional alignment methods such as FASTA or BLAST, which are still widely used for MR. In addition, by designing MR pipelines that integrate phasing and automated refinement and allow parallel processing of such calculations, one can effectively increase the success rate of MR. Here, updated results from the JCSG MR pipeline are presented, which to date has solved 33 MR structures with less than 35% sequence identity to the closest homologue of known structure. By using difficult MR problems as examples, it is demonstrated that successful MR phasing is possible even in cases where the similarity between the model and the template can only be detected with fold-recognition algorithms. In the first step, several search models are built based on all homologues found in the PDB by fold-recognition algorithms. The models resulting from this process are used in parallel MR searches with different combinations of input parameters of the MR phasing algorithm. The putative solutions are subjected to rigid-body and restrained crystallographic refinement and ranked based on the final values of free R factor, figure of merit and deviations from ideal geometry. Finally, crystal packing and electron-density maps are checked to identify the correct solution. If this procedure does not yield a solution with interpretable electron-density maps, then even more alternative models are prepared. The structurally variable regions of a protein family are identified based on alignments of sequences and known structures from that family and appropriate trimmings of the models are proposed. All combinations of these trimmings are applied to the search models and the resulting set of models is used in the MR pipeline. It is estimated that with the improvements in model building and exhaustive parallel searches with existing phasing algorithms, MR can be successful for more than 50% of recognizable homologues of known structures below the threshold of 35% sequence identity. This implies that about one-third of the proteins in a typical bacterial proteome are potential MR targets.

Structure of the human glucagon class B G-protein-coupled receptor

“…The “mixed model” templates were then superimposed together and manually trimmed to remove structurally poorly conserved regions. Parallel MR searches with Phaser were then carried out using these GPCR models along with the high resolution BRIL structure (PDB 1M6T) as search models on a linux cluster 53 . The search template based on rhodopsin (PDB 2Z73) produced a potential solution (TFZ=8.5).…”

Structural Basis of Membrane Targeting by the Dock180 Family of Rho Family Guanine Exchange Factors (Rho-GEFs)

“…Consequently, a more extensive molecular replacement search strategy was pursued using the search pipeline (35) based on MOLREP (36), with automated parameter-space screening. The search employed models derived from the distant homology sequence-recognition algorithm (37).…”

Improvement of molecular-replacement models withSculptor

“…It frequently happens that a solution has been found but it is not possible to identify it from the search statistics. In such a case, automated rebuilding calculations may identify one of the candidates as a true solution (Keegan & Winn, 2007;Long et al, 2008;Schwarzenbacher et al, 2008). Visual inspection of the model in the electron density is rarely helpful, as nonsolutions can also give very clear (but incorrect) maps.…”

“…To some extent, the powerful but computationally intensive combinatorial trimming technique described by Schwarzenbacher et al (2008) can be approximated by this approach. It is not clear how many different protocols are necessary to obtain optimal performance, but a moderate number seems to give good results.…”