Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas, 2030 Becker Dr, Lawrence, KS 66047, USA

Abstract

Background

Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions.

Results

An on-line version of I-TASSER is developed at the KU Center for Bioinformatics which has generated protein structure predictions for thousands of modeling requests from more than 35 countries. A scoring function (C-score) based on the relative clustering structural density and the consensus significance score of multiple threading templates is introduced to estimate the accuracy of the I-TASSER predictions. A large-scale benchmark test demonstrates a strong correlation between the C-score and the TM-score (a structural similarity measurement with values in [0, 1]) of the first models with a correlation coefficient of 0.91. Using a C-score cutoff > -1.5 for the models of correct topology, both false positive and false negative rates are below 0.1. Combining C-score and protein length, the accuracy of the I-TASSER models can be predicted with an average error of 0.08 for TM-score and 2 Å for RMSD.

Conclusion

The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I-TASSER models. The output of the I-TASSER server for each query includes up to five full-length models, the confidence score, the estimated TM-score and RMSD, and the standard deviation of the estimations. The I-TASSER server is freely available to the academic community at

Background

Protein structure prediction refers to the effort of generating 3-dimensional models from amino acid sequences using computer algorithms. However, structure modeling processes often involve human interventions because the human-expert knowledge combined with biochemical information (function, mutagenesis, catalytic residues, etc.) could help in both structural assembly and model selection

Since the first public release in November 2006, the I-TASSER server has generated structure predictions for thousands of modeling requests from various laboratories in the world. We have been frequently asked by the users about how the quality of the I-TASSER models should be annotated because this will essentially decide how they will exploit the predictions in their research. The general idea of the modeling quality estimation of 3D models has been pursued by a number of authors

In this work, we introduce the on-line setting of the I-TASSER server and develop a confidence scoring system which can provide the users with a simple and reliable assessment of the I-TASSER models. Different from most of the MQAP programs that assess models purely based on the structure of the final models, the confidence scoring function developed here incorporates the information and parameters of the modeling simulations.

Implementation

I-TASSER method

I-TASSER is a hierarchical protein structure modeling approach based on the secondary-structure enhanced Profile-Profile threading Alignment (PPA)

The target sequences are first threaded through a representative PDB structure library (with a pair-wise sequence identity cut-off of 70%) to search for the possible folds by four simple variants of PPA methods, with different combinations of the hidden Markov model _{α }atoms and the side-chain centers of mass specified. Pulchra

If any region with >80 residues has no aligned residues in at least two strong PPA alignments of Z-score > Z_{0 }(see below), the target will be judged as a multiple domain protein and domain boundaries are automatically assigned based on the borders of the large gaps. I-TASSER simulations will be run for the full chain as well as the separate domains. The final full-length models are generated by docking the model of domains together. The domain docking is performed by a quick Metropolis Monte Carlo simulation where the energy is defined as the RMSD of domain models to the full-chain model plus the reciprocal of the number of steric clashes between domains. The goal of the docking is to find the domain orientation that is closest to the I-TASSER full-chain model but has the minimum steric clashes. This procedure does not influence the multiple domain proteins which have all domains completely aligned by the PPAs.

C-score

The C-score of the I-TASSER models is defined as

where M is the multiplicity of structures in the SPICKER cluster; M_{tot }is the total number of the I-TASSER structure decoys used in the clustering; ⟨RMSD⟩ is the average RMSD of the decoys to the cluster centroid; Z(_{0}(_{0}(1) = 7.0, Z_{0}(2) = 8.5, Z_{0}(3) = 8.0, Z_{0}(4) = 10.5.

The first two factors of Equation 1 account for the degree of structure convergence in the SPICKER clustering, which correlates with the consistency of the external restraints and the inherent I-TASSER potential. The third factor accounts for the quality of threading alignments. The logarithm in Equation 1 is to adjust the C-score values in an approximately even distribution. A previously defined C-score has been shown to have a strong correlation with the quality of the predicted models _{0 }is used instead of the Z-score itself which makes it easy to extend the definition to the cases when templates are generated by different threading algorithms. Second, it accounts for the consensus of alignment confidence of multiple threading programs rather than one threading program.

We also tried other alternatives for the C-score definition. For example, if we add ⟨TM-score⟩, the average TM-score of the decoys to the cluster centroid, in the numerator of the second factor in Equation 1, the correlation between the C-score and TM-score will increase by ~2%. But it does not increase the correlation of C-score with RMSD and the calculation of ⟨TM-score⟩ will increase the SPICKER running time by ~20%. So we did not include ⟨TM-score⟩ in the C-score definition. We also attempted to optimize the powers of the three factors of Equation 1 by maximizing the correlation between C-score and the quality of final models in the training proteins. Interestingly, the optimized powers of all three factors are close to 1, which indicates that the C-score in Equation 1 is close to an optimal definition if considering these 3 factors.

TM-score

TM-score is defined to assess the topological similarity of two protein structures

where _{i }is the distance of the

Server setting

The URL address of the on-line I-TASSER server is listed at the end of the paper. To use the server, what users need to provide is the amino acid sequence of the proteins to be modeled in the FASTA format. Currently, the acceptable size range of the targets is between 10–1,500 residues. Depending on the protein size, the I-TASSER modeling procedure takes a maximum of 48 hours (typically 5–10 hours for a sequence around 200 residues). After the modeling is finished, an email will be sent to the users, which include the PDB format files of up to 5 predicted models, C-score of the models, and the predicted RMSD and TM-score of the first model. A brief explanation of the RMSD, TM-score, and C-score is also provided in the email.

Once a prediction is made, a GIF visual file is made for each of the I-TASSER models so that the users can get a quick on-line view of how the topology of their models looks like. The PDB files and the visual files are kept on our server for 365 days and made publicly downloadable at

Results and Discussions

For the benchmark of the I-TASSER server, we collect 800 nonhomologous single-domain proteins directly from the PDB library

It should be mentioned that here we benchmark the I-TASSER algorithm only on the single-domain proteins. For multiple-domain proteins, a small misorientation of the domains may result in dramatic change in TM-score and RMSD values even if the topology of the individual domains is unchanged, which can result in divergent correlations of the C-score and the overall model qualities. Consequently, the confidence score and quality estimation of multiple-domain models should be understood approximately as those for the individual domain units.

Correlation of C-score and model qualities

In Figure

TM-score (a) and RMSD (b) versus C-score of the I-TASSER models for 500 testing proteins

TM-score (a) and RMSD (b) versus C-score of the I-TASSER models for 500 testing proteins. The dashed curve in (a) is from Equation 3 which is fit from the 300 training proteins and used for estimating the TM-score of the I-TASSER models. The solid circles are the root mean squared deviation from the estimated TM-score values (RMSTD). The solid curve is from Equation 4 which is fit from the 300 training proteins. The dotted lines are the TM-score and C-score cutoffs for correct folds.

The correlation of RMSD with the C-score is not as strong as that of the TM-score (Figure

Two examples of the I-TASSER models from 1ca4A and 1cmaA

Two examples of the I-TASSER models from 1ca4A and 1cmaA. Both models have similar RMSD values but indicate significantly different modeling qualities. In the superposition, the thin backbones are the native structure and thick backbones the I-TASSER models. Blue to red runs from N- to C-terminal.

The second reason for the low RMSD/C-score correlation is due to the inherent size dependence of RMSD. In Figure

TM-score (a) and RMSD (b) of the I-TASSER models versus the length of target proteins

TM-score (a) and RMSD (b) of the I-TASSER models versus the length of target proteins. The numbers indicate the Pearson correlation coefficients.

In Figure

As a control, we also calculate the correlation of TM-score (or RMSD) with the sequence identity between the target and the best template, which is 0.33 (or -0.23). The low correlation is not surprising because all homologous templates with a high sequence identity >30% have been excluded and the profile-profile programs often identify templates of correct topology even when the sequence identity to the target is low.

RMSD versus C-score-ln(

RMSD versus C-score-ln(

Quantitative estimate of the quality of I-TASSER models

Based on the I-TASSER models of the 300 training proteins, we fit a two-order polynomial to the TM-score/C-score data by the least square fitting method

^{2 }+ 0.13*C-score + 0.71,

with a root mean squared TM-score deviation (RMSTD) of 0.08 for the training protein set. In Figure ^{2}⟩ and the average error of estimation is ⟨|TM-score - ⟨TM-score⟩|⟩, where ⟨TM-score⟩ is the average TM-score in the training set and the estimated TM-score in the test set. If we use RMSTD as the standard deviation of the TM-score estimation, there is a probability of 68.3% that the real TM-score will fall in the range of TM-score ± RMSTD

In the lower part of Figure

An overlap of Equation 4 with the RMSTD data is shown in Figure

Since the RMSD of the I-TASSER models correlates better with C-score-ln(

^{2 }- 1.14(C-score - ln

with a root mean squared RMSD deviation (RMSRD) of 3.1 Å. In Figure

In the low part of Figure

Conclusion

We develop the I-TASSER server for the automated full-length protein structure prediction. A series of accessorial WebPages are designed to facilitate the users in submitting, viewing and tracking the predictions. Based on the statistical significance of the PPA threading alignments and the structure convergence of the Monte Carlo simulations, a new confidence score (C-score) is introduced and benchmarked for the I-TASSER server, which demonstrates a strong correlation with the real quality of the final models. The Pearson correlation coefficients of the C-score with TM-score and RMSD are 0.91 and 0.75 respectively. The strong correlation data allows us to make quantitative estimates of the accuracy of the I-TASSER predictions. Using a 2-order polynomial equation fit from 300 training proteins, we can predict the TM-score and RMSD of the final models with an average error of 0.08 and 2.0 Å respectively in a large scale benchmark test.

For each submitted sequence, following items will be returned to the users by email after the I-TASSER modeling: (1) up to five predicted models ranked based on the structure density of the SPICKER clustering; (2) C-score of all the I-TASSER models; (3) estimated TM-score and RMSD for the first model in the form of

It should be mentioned that the estimated qualities are provided only for the first model, although for the purpose of providing more information the C-score of all 5 models are sent to the users. The correlation of C-score and modeling quality for the lower-rank models is much weaker than that for the first model. This is understandable because the conformational space covered by the I-TASSER simulations is limited. For easy targets almost all decoys are near-native and the structures are mainly clustered in the first cluster. After removing the structures in the first cluster, the size of the lower-rank clusters will be much smaller which may be comparable to that of hard targets. But the quality of the lower-rank clusters from the easy targets is still on average better than that from the hard targets because most decoys generated in the hard targets are incorrect. Nevertheless, there is a correlation between the rank and the quality of the clusters for the same target. In this set of test proteins, the average TM-score (RMSD) of the top-five models are 0.501 (9.6 Å), 0.468 (10.6 Å), 0.466 (10.7 Å), 0.461 (11.1 Å), and 0.454 (11.3 Å) respectively. Therefore, the C-score and predicted data should be considered as an upper-limit estimate for the quality of all I-TASSER models.

Availability and requirements

Project name: I-TASSER server

Project home page:

Operating system(s): Windows, Linux, Mac

Programming language: Perl, Fortran77

License: GPL

Any restrictions to use by non-academics: license needed

Abbreviations

I-TASSER: iterative threading assembly refinement algorithm.

PPA: profile-profile alignment threading algorithm.

RMSD: root mean squared deviation.

RMSRD: root mean squared RMSD deviation from average or estimated RMSD.

RMSTD: root mean squared TM-score deviation from average or estimated TM-score.

Authors' contributions

YZ developed the I-TASSER server, performed the benchmark calculation and wrote the manuscript. He has read and approved the final manuscript.

Acknowledgements

The author wants to thank Dr. Sitao Wu for help in constructing the WebPages. The project is partly supported by KU Start-up Fund 06194.