Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins

Abstract

Molecular replacement (MR) often requires templates with high homology to solve the phase problem in X-ray crystallography. I-TASSER-MR has been developed to test whether the success rate for structure determination of distant-homology proteins could be improved by a combination of iterative fragmental structure-assembly simulations with progressive sequence truncation designed to trim regions with high variation. The pipeline was tested on two independent protein sets consisting of 61 proteins from CASP8 and 100 high-resolution proteins from the PDB. After excluding homologous templates, I-TASSER generated full-length models with an average TM-score of 0.773, which is 12% higher than the best threading templates. Using these as search models, I-TASSER-MR found correct MR solutions for 95 of 161 targets as judged by having a TFZ of >8 or with the final structure closer to the native than the initial search models. The success rate was 16% higher than when using the best threading templates. I-TASSER-MR was also applied to 14 protein targets from structure genomics centers. Seven of these were successfully solved by I-TASSER-MR. These results confirm that advanced structure assembly and progressive structural editing can significantly improve the success rate of MR for targets with distant homology to proteins of known structure.

Keywords: I-TASSER; X-ray crystallography; molecular replacement; protein structure prediction; threading.

Figure 1

Flow chart of I-TASSER-MR. The target sequence is first threaded through nonredundant structures from the PDB library to identify structure templates (column 1), with three-dimensional full-length models constructed by iterative fragment-reassembly simulations (column 2). The structure models are progressively edited based on AVS, which demarks poorly predicted regions (column 3), and the resulting models are used in a standard MR search by Phaser followed by automated model building and refinement with phenix.autobuild (column 4).

Figure 2

Illustration of progressive truncation in I-TASSER-MR. (a) Superposition of the initial I-TASSER model (blue) and the experimental structure (cyan; PDB entry 3d89). The structure regions with a high AVS score selected for truncation by I-TASSER-MR are marked in red. The r.m.s.d., PBS and GDT-TS score of the two structures were 2.76 Å (821 atoms), 1.78 Å (821 atoms) and 0.66, respectively. (b) Overlay of the I-TASSER-MR solution (blue) generated without model truncation on the experimental 2m|F _o| − D|F _c| σ_A-weighted map calculated by the EDS contoured at 2σ. (c) Superposition of the final I-TASSER-MR structure solved using model truncation (blue) and the experimental structure. The r.m.s.d., PBS and GDT-TS scores of the structure were 0.95 Å (809 atoms), 0.24 Å (809 atoms) and 0.74, respectively. (d) Overlay of the successful truncated I-TASSER-MR structure (blue) and the experimental electron density.

Figure 3

Impact of B-factor assignment on MR solutions. (a) Superposition of PDB entry 2tnf (yellow) with I-TASSER-MR-generated solutions with constant (green, left panel) or AVS-based (red, right panel) B-factor schemes, which are overlaid on a 2m|F _o| − D|F _c| σ_A-weighted map calculated by the EDS contoured at 2σ. (b) Superposition of the native structure of PDB entry 3dcp (yellow) with I-TASSER-MR-generated solutions with a constant (green, left panel) or ASA-based (red, right panel) B-factor setting, which are overlaid on the corresponding electron-density map.

Figure 4

Typical distribution of residue truncations. (a) Superposition of the initial I-TASSER model (blue) and the native structure (cyan) of PDB entry 3dfa. Truncated residues are highlighted in red for the initial I-TASSER model; the analogous residues in the deposited structure are in green. The r.m.s.d., PBS and GDT-TS scores of the two structures are 2.98 Å (1761 atoms), 2.36 Å (1761 atoms) and 0.68, respectively. (b) Superposition of the I-TASSER-MR model and the experimental structure. The r.m.s.d., PBS and GDT-TS score of the two structures are 0.74 Å (1430 atoms), 0.24 Å (1430 atoms) and 0.63, respectively. (c) Overlay of the I-TASSER-MR structure (blue) on the 2m|F _o| − D|F _c| σ_A-weighted map contoured at 2σ.

Figure 5

Success rate of I-TASSER-MR at different levels of truncation. The x axis indicates the percentage of residues in the search model after model truncation, normalized by the target length.

Figure 6

Scatter plots of the resolution of the diffraction data for the 95 successful protein targets. The x axis shows the percentage of the sequence remaining in the search models for the successful MR solution. Each point represents one successful solution from a target protein at a specific truncation, whereby one protein can have multiple successful MR solutions at different truncations.

Figure 7

Correlation of MR with the C-score of the I-TASSER models. Histogram of the C-score of the I-TASSER models (light bars) and the average success rate of I-TASSER-MR in each C-score range (dark bars).