MR-REX: molecular replacement by cooperative conformational search and occupancy optimization on low-accuracy protein models.

Molecular replacement (MR) has commonly been employed to derive the phase information in protein crystal X-ray diffraction, but its success rate decreases rapidly when the search model is dissimilar to the target. MR-REX has been developed to perform an MR search by replica-exchange Monte Carlo simulations, which enables cooperative rotation and translation searches and simultaneous clash and occupancy optimization. MR-REX was tested on a set of 1303 protein structures of different accuracies and successfully placed 699 structures at positions that have an r.m.s.d. of below 2 Što the target position, which is 10% higher than was obtained by Phaser. However, cases studies show that many of the models for which Phaser failed and MR-REX succeeded can be solved by Phaser by pruning them and using nondefault parameters. The factors effecting success and the parts of the methodology which lead to success are studied. The results demonstrate a new avenue for molecular replacement which outperforms (and has results that are complementary to) the state-of-the-art MR methods, in particular for distantly homologous proteins.

I-TASSER-MR: automated molecular replacement for distant-homology proteins using iterative fragment assembly and progressive sequence truncation.

Molecular replacement (MR) is one of the most common techniques used for solving the phase problem in X-ray crystal diffraction. The success rate of MR however drops quickly when the sequence identity between query and templates is reduced, while the I-TASSER-MR server is designed to solve the phase problem for proteins that lack close homologous templates. Starting from a sequence, it first generates full-length models using I-TASSER by iterative structural fragment reassembly. A progressive sequence truncation procedure is then used for editing the models based on local variations of the structural assembly simulations. Next, the edited models are submitted to MR-REX to search for optimal placements in the crystal unit-cells through replica-exchange Monte Carlo simulations, with the phasing results used by CNS for final atomic model refinement and selection. The I-TASSER-MR algorithm was tested in large-scale benchmark datasets and solved 36% more targets compared to using the best threading templates. The server takes primary sequence and raw crystal diffraction data as input, with output containing annotated phase information and refined structure models. It also allows users to choose between different methods for setting B-factors and the number of models used for phasing. The online server is freely available at http://zhanglab.ccmb.med.umich.edu/I-TASSER-MR.

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

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.

Integration of QUARK and I-TASSER for Ab Initio Protein Structure Prediction in CASP11.

We tested two pipelines developed for template-free protein structure prediction in the CASP11 experiment. First, the QUARK pipeline constructs structure models by reassembling fragments of continuously distributed lengths excised from unrelated proteins. Five free-modeling (FM) targets have the model successfully constructed by QUARK with a TM-score above 0.4, including the first model of T0837-D1, which has a TM-score = 0.736 and RMSD = 2.9 Å to the native. Detailed analysis showed that the success is partly attributed to the high-resolution contact map prediction derived from fragment-based distance-profiles, which are mainly located between regular secondary structure elements and loops/turns and help guide the orientation of secondary structure assembly. In the Zhang-Server pipeline, weakly scoring threading templates are re-ordered by the structural similarity to the ab initio folding models, which are then reassembled by I-TASSER based structure assembly simulations; 60% more domains with length up to 204 residues, compared to the QUARK pipeline, were successfully modeled by the I-TASSER pipeline with a TM-score above 0.4. The robustness of the I-TASSER pipeline can stem from the composite fragment-assembly simulations that combine structures from both ab initio folding and threading template refinements. Despite the promising cases, challenges still exist in long-range beta-strand folding, domain parsing, and the uncertainty of secondary structure prediction; the latter of which was found to affect nearly all aspects of FM structure predictions, from fragment identification, target classification, structure assembly, to final model selection. Significant efforts are needed to solve these problems before real progress on FM could be made. Proteins 2016; 84(Suppl 1):76-86. © 2015 Wiley Periodicals, Inc.

Template-based protein structure prediction in CASP11 and retrospect of I-TASSER in the last decade.

We report the structure prediction results of a new composite pipeline for template-based modeling (TBM) in the 11th CASP experiment. Starting from multiple structure templates identified by LOMETS based meta-threading programs, the QUARK ab initio folding program is extended to generate initial full-length models under strong constraints from template alignments. The final atomic models are then constructed by I-TASSER based fragment reassembly simulations, followed by the fragment-guided molecular dynamic simulation and the MQAP-based model selection. It was found that the inclusion of QUARK-TBM simulations as an intermediate modeling step could help improve the quality of the I-TASSER models for both Easy and Hard TBM targets. Overall, the average TM-score of the first I-TASSER model is 12% higher than that of the best LOMETS templates, with the RMSD in the same threading-aligned regions reduced from 5.8 to 4.7 Å. Nevertheless, there are nearly 18% of TBM domains with the templates deteriorated by the structure assembly pipeline, which may be attributed to the errors of secondary structure and domain orientation predictions that propagate through and degrade the procedures of template identification and final model selections. To examine the record of progress, we made a retrospective report of the I-TASSER pipeline in the last five CASP experiments (CASP7-11). The data show no clear progress of the LOMETS threading programs over PSI-BLAST; but obvious progress on structural improvement relative to threading templates was witnessed in recent CASP experiments, which is probably attributed to the integration of the extended ab initio folding simulation with the threading assembly pipeline and the introduction of atomic-level structure refinements following the reduced modeling simulations. Proteins 2016; 84(Suppl 1):233-246. © 2015 Wiley Periodicals, Inc.

Modeling the hydration layer around proteins: applications to small- and wide-angle x-ray scattering.

Small-/wide-angle x-ray scattering (SWAXS) experiments can aid in determining the structures of proteins and protein complexes, but success requires accurate computational treatment of solvation. We compare two methods by which to calculate SWAXS patterns. The first approach uses all-atom explicit-solvent molecular dynamics (MD) simulations. The second, far less computationally expensive method involves prediction of the hydration density around a protein using our new HyPred solvation model, which is applied without the need for additional MD simulations. The SWAXS patterns obtained from the HyPred model compare well to both experimental data and the patterns predicted by the MD simulations. Both approaches exhibit advantages over existing methods for analyzing SWAXS data. The close correspondence between calculated and observed SWAXS patterns provides strong experimental support for the description of hydration implicit in the HyPred model.

Printing of polymer microcapsules for enzyme immobilization on paper substrate.

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.

Modeling the hydration layer around proteins: HyPred.

Protein hydration plays an integral role in determining protein function and stability. We develop a simple method with atomic level precision for predicting the solvent density near the surface of a protein. A set of proximal radial distribution functions are defined and calculated for a series of different atom types in proteins using all-atom, explicit solvent molecular dynamic simulations for three globular proteins. A major improvement in predicting the hydration layer is found when the protein is held immobile during the simulations. The distribution functions are used to develop a model for predicting the hydration layer with sub-1-Angstrom resolution without the need for additional simulations. The model and the distribution functions for a given protein are tested in their ability to reproduce the hydration layer from the simulations for that protein, as well as those for other proteins and for simulations in which the protein atoms are mobile. Predictions for the density of water in the hydration shells are then compared with high occupancy sites observed in crystal structures. The accuracy of both tests demonstrates that the solvation model provides a basis for quantitatively understanding protein solvation and thereby predicting the hydration layer without additional simulations.