Molecular replacement using structure predictions from databases

Abstract

Molecular replacement (MR) is the predominant route to solution of the phase problem in macromolecular crystallography. Where the lack of a suitable homologue precludes conventional MR, one option is to predict the target structure using bioinformatics. Such modelling, in the absence of homologous templates, is called ab initio or de novo modelling. Recently, the accuracy of such models has improved significantly as a result of the availability, in many cases, of residue-contact predictions derived from evolutionary covariance analysis. Covariance-assisted ab initio models representing structurally uncharacterized Pfam families are now available on a large scale in databases, potentially representing a valuable and easily accessible supplement to the PDB as a source of search models. Here, the unconventional MR pipeline AMPLE is employed to explore the value of structure predictions in the GREMLIN and PconsFam databases. It was tested whether these deposited predictions, processed in various ways, could solve the structures of PDB entries that were subsequently deposited. The results were encouraging: nine of 27 GREMLIN cases were solved, covering target lengths of 109-355 residues and a resolution range of 1.4-2.9 Å, and with target-model shared sequence identity as low as 20%. The cluster-and-truncate approach in AMPLE proved to be essential for most successes. For the overall lower quality structure predictions in the PconsFam database, remodelling with Rosetta within the AMPLE pipeline proved to be the best approach, generating ensemble search models from single-structure deposits. Finally, it is shown that the AMPLE-obtained search models deriving from GREMLIN deposits are of sufficiently high quality to be selected by the sequence-independent MR pipeline SIMBAD. Overall, the results help to point the way towards the optimal use of the expanding databases of ab initio structure predictions.

Keywords: ab initio modelling; ab initio structure predictions; databases; molecular replacement.

Figure 1

Flowchart showing the methods used to treat search models obtained from GREMLIN and PconsFam prior to AMPLE or AMPLE single-model mode. The relative success of each method is represented in green, orange or red, where green represents a more successful method and red represents a less successful method.

Figure 2

(a) The 30 models obtained from the GREMLIN database for PF01790 (magenta) aligned with the crystallized structure, PDB entry 5azb (rainbow from blue at the N-­terminus to red at the C-terminus). (b) The best-performing AMPLE-derived ensemble (magenta), derived by truncating cluster 1 down to 12% (33 residues), aligned with the crystallized structure, PDB entry 5azb (rainbow). (c) The 30 models obtained from the GREMLIN database for PF02470 (magenta) aligned with the crystallized structure, PDB entry 5uw2 (rainbow). (d) The best-performing AMPLE-derived ensemble (magenta), derived by truncating cluster 2 down to 80% (96 residues), aligned with the crystal structure, PDB entry 5uw2 (rainbow). (e) The 30 models obtained from the GREMLIN database for PF03883 (magenta) aligned with the crystallized structure, PDB entry 5caj (rainbow). (f) The best-performing AMPLE-derived ensemble (magenta), derived by truncating cluster 1 down to 54% (137 residues), aligned with the crystallized structure, PDB entry 5caj (rainbow). (g) The 30 models obtained from the GREMLIN database for PF06130 (magenta) aligned with the crystallized structure, PDB entry 5cuo (rainbow). (h) The best-performing AMPLE-derived ensemble (magenta), derived by truncating cluster 3 down to 70% (138 residues), aligned with the crystallized structure, PDB entry 5cuo (rainbow).

Figure 3

(a) PconsFam model for PF02660 (magenta) aligned with the crystallized structure, PDB entry 5jx5 (rainbow). (b) An untruncated AMPLE ensemble (magenta ribbon), following Rosetta remodelling, aligned with the crystallized structure, PDB entry 5jx5 (rainbow). (c) The truncated AMPLE ensemble (c1_23_r3_polyAla) obtained from the Rosetta-remodelled versions of the PconsFam model for PF02660 (magenta) aligned with the crystallized structure, PDB entry 5jx5 (rainbow).

Figure 4

(a) PconsFam model for PF00071 (magenta) aligned with the crystallized structure, PDB entry 1yzq (rainbow). (b) An untruncated AMPLE ensemble (magenta ribbon), following CONCOORD, aligned with the crystallized structure, PDB entry 1yzq (rainbow). (c) The AMPLE ensemble obtained from the CONCOORD derivatives for PF00071 (magenta) aligned with the crystallized structure, PDB entry 1yzq (rainbow).

Figure 5

Cross-eyed stereoview of the AMPLE ensemble (c1_t74_r3_polyAla) which gave the best score in the SIMBAD search for PF09819 (magenta) aligned with the crystallized structure, PDB entry 5edl (rainbow from blue at the N-terminus to red at the C-terminus).