EquiPNAS: improved protein-nucleic acid binding site prediction using protein-language-model-informed equivariant deep graph neural networks.

Protein language models (pLMs) trained on a large corpus of protein sequences have shown unprecedented scalability and broad generalizability in a wide range of predictive modeling tasks, but their power has not yet been harnessed for predicting protein-nucleic acid binding sites, critical for characterizing the interactions between proteins and nucleic acids. Here, we present EquiPNAS, a new pLM-informed E(3) equivariant deep graph neural network framework for improved protein-nucleic acid binding site prediction. By combining the strengths of pLM and symmetry-aware deep graph learning, EquiPNAS consistently outperforms the state-of-the-art methods for both protein-DNA and protein-RNA binding site prediction on multiple datasets across a diverse set of predictive modeling scenarios ranging from using experimental input to AlphaFold2 predictions. Our ablation study reveals that the pLM embeddings used in EquiPNAS are sufficiently powerful to dramatically reduce the dependence on the availability of evolutionary information without compromising on accuracy, and that the symmetry-aware nature of the E(3) equivariant graph-based neural architecture offers remarkable robustness and performance resilience. EquiPNAS is freely available at https://github.com/Bhattacharya-Lab/EquiPNAS.

lociPARSE: a locality-aware invariant point attention model for scoring RNA 3D structures.

A scoring function that can reliably assess the accuracy of a 3D RNA structural model in the absence of experimental structure is not only important for model evaluation and selection but also useful for scoring-guided conformational sampling. However, high-fidelity RNA scoring has proven to be difficult using conventional knowledge-based statistical potentials and currently-available machine learning-based approaches. Here we present lociPARSE, a locality-aware invariant point attention architecture for scoring RNA 3D structures. Unlike existing machine learning methods that estimate superposition-based root mean square deviation (RMSD), lociPARSE estimates Local Distance Difference Test (lDDT) scores capturing the accuracy of each nucleotide and its surrounding local atomic environment in a superposition-free manner, before aggregating information to predict global structural accuracy. Tested on multiple datasets including CASP15, lociPARSE significantly outperforms existing statistical potentials (rsRNASP, cgRNASP, DFIRE-RNA, and RASP) and machine learning methods (ARES and RNA3DCNN) across complementary assessment metrics. lociPARSE is freely available at https://github.com/Bhattacharya-Lab/lociPARSE.

EquiPNAS: improved protein-nucleic acid binding site prediction using protein-language-model-informed equivariant deep graph neural networks.

Protein language models (pLMs) trained on a large corpus of protein sequences have shown unprecedented scalability and broad generalizability in a wide range of predictive modeling tasks, but their power has not yet been harnessed for predicting protein-nucleic acid binding sites, critical for characterizing the interactions between proteins and nucleic acids. Here we present EquiPNAS, a new pLM-informed E(3) equivariant deep graph neural network framework for improved protein-nucleic acid binding site prediction. By combining the strengths of pLM and symmetry-aware deep graph learning, EquiPNAS consistently outperforms the state-of-the-art methods for both protein-DNA and protein-RNA binding site prediction on multiple datasets across a diverse set of predictive modeling scenarios ranging from using experimental input to AlphaFold2 predictions. Our ablation study reveals that the pLM embeddings used in EquiPNAS are sufficiently powerful to dramatically reduce the dependence on the availability of evolutionary information without compromising on accuracy, and that the symmetry-aware nature of the E(3) equivariant graph-based neural architecture offers remarkable robustness and performance resilience. EquiPNAS is freely available at https://github.com/Bhattacharya-Lab/EquiPNAS.

E(3) equivariant graph neural networks for robust and accurate protein-protein interaction site prediction.

Artificial intelligence-powered protein structure prediction methods have led to a paradigm-shift in computational structural biology, yet contemporary approaches for predicting the interfacial residues (i.e., sites) of protein-protein interaction (PPI) still rely on experimental structures. Recent studies have demonstrated benefits of employing graph convolution for PPI site prediction, but ignore symmetries naturally occurring in 3-dimensional space and act only on experimental coordinates. Here we present EquiPPIS, an E(3) equivariant graph neural network approach for PPI site prediction. EquiPPIS employs symmetry-aware graph convolutions that transform equivariantly with translation, rotation, and reflection in 3D space, providing richer representations for molecular data compared to invariant convolutions. EquiPPIS substantially outperforms state-of-the-art approaches based on the same experimental input, and exhibits remarkable robustness by attaining better accuracy with predicted structural models from AlphaFold2 than what existing methods can achieve even with experimental structures. Freely available at https://github.com/Bhattacharya-Lab/EquiPPIS, EquiPPIS enables accurate PPI site prediction at scale.

The transformative power of transformers in protein structure prediction.

Transformer neural networks have revolutionized structural biology with the ability to predict protein structures at unprecedented high accuracy. Here, we report the predictive modeling performance of the state-of-the-art protein structure prediction methods built on transformers for 69 protein targets from the recently concluded 15th Critical Assessment of Structure Prediction (CASP15) challenge. Our study shows the power of transformers in protein structure modeling and highlights future areas of improvement.

PIQLE: protein-protein interface quality estimation by deep graph learning of multimeric interaction geometries.

Motivation: Accurate modeling of protein-protein interaction interface is essential for high-quality protein complex structure prediction. Existing approaches for estimating the quality of a predicted protein complex structural model utilize only the physicochemical properties or energetic contributions of the interacting atoms, ignoring evolutionarily information or inter-atomic multimeric geometries, including interaction distance and orientations. Results: Here, we present PIQLE, a deep graph learning method for protein-protein interface quality estimation. PIQLE leverages multimeric interaction geometries and evolutionarily information along with sequence- and structure-derived features to estimate the quality of individual interactions between the interfacial residues using a multi-head graph attention network and then probabilistically combines the estimated quality for scoring the overall interface. Experimental results show that PIQLE consistently outperforms existing state-of-the-art methods including DProQA, TRScore, GNN-DOVE and DOVE on multiple independent test datasets across a wide range of evaluation metrics. Our ablation study and comparison with the self-assessment module of AlphaFold-Multimer repurposed for protein complex scoring reveal that the performance gains are connected to the effectiveness of the multi-head graph attention network in leveraging multimeric interaction geometries and evolutionary information along with other sequence- and structure-derived features adopted in PIQLE. Availability and implementation: An open-source software implementation of PIQLE is freely available at https://github.com/Bhattacharya-Lab/PIQLE. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

iQDeep: an integrated web server for protein scoring using multiscale deep learning models.

The remarkable recent advances in protein structure prediction have enabled computational modeling of protein structures with considerably higher accuracy than ever before. While state-of-the-art structure prediction methods provide self-assessment confidence scores of their own predictions, an independent and open-access system for protein scoring is still needed that can be applied to a broad range of predictive modeling scenarios. Here, we present iQDeep, an integrated and highly customizable web server for protein scoring, freely available at http://fusion.cs.vt.edu/iQDeep. The underlying method of iQDeep employs multiscale deep residual neural networks (ResNets) to perform residue-level error classifications, and then probabilistically combines the error classifications for protein scoring. By adjusting the error resolutions, our method can reliably estimate the standard- or high-accuracy variants of the Global Distance Test metric for versatile protein scoring. The performance of the method has been extensively tested and compared against the state-of-the-art approaches in multiple rounds of Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments including benchmark assessment in CASP12 and CASP13 as well as blind evaluation in CASP14. The iQDeep web server offers a number of convenient features, including (i) the choice of individual and batch processing modes; (ii) an interactive and privacy-preserving web interface for automated job submission, tracking, and results retrieval; (iii) web-based quantitative and visual analyses of the results including overall estimated score and its residue-wise breakdown along with agreements between various sequence- and structural-level features; (iv) extensive help information on job submission and results interpretation via web-based tutorial and help tooltips.

Contact-Assisted Threading in Low-Homology Protein Modeling.

The ability to successfully predict the three-dimensional structure of a protein from its amino acid sequence has made considerable progress in the recent past. The progress is propelled by the improved accuracy of deep learning-based inter-residue contact map predictors coupled with the rising growth of protein sequence databases. Contact map encodes interatomic interaction information that can be exploited for highly accurate prediction of protein structures via contact map threading even for the query proteins that are not amenable to direct homology modeling. As such, contact-assisted threading has garnered considerable research effort. In this chapter, we provide an overview of existing contact-assisted threading methods while highlighting the recent advances and discussing some of the current limitations and future prospects in the application of contact-assisted threading for improving the accuracy of low-homology protein modeling.

PIQLE: protein-protein interface quality estimation by deep graph learning of multimeric interaction geometries.

Accurate modeling of protein-protein interaction interface is essential for high-quality protein complex structure prediction. Existing approaches for estimating the quality of a predicted protein complex structural model utilize only the physicochemical properties or energetic contributions of the interacting atoms, ignoring evolutionarily information or inter-atomic multimeric geometries, including interaction distance and orientations. Here we present PIQLE, a deep graph learning method for protein-protein interface quality estimation. PIQLE leverages multimeric interaction geometries and evolutionarily information along with sequence- and structure-derived features to estimate the quality of the individual interactions between the interfacial residues using a multihead graph attention network and then probabilistically combines the estimated quality of the interfacial residues for scoring the overall interface. Experimental results show that PIQLE consistently outperforms existing state-of-the-art methods on multiple independent test datasets across a wide range of evaluation metrics. Our ablation study reveals that the performance gains are connected to the effectiveness of the multihead graph attention network in leveraging multimeric interaction geometries and evolutionary information along with other sequence- and structure-derived features adopted in PIQLE. An open-source software implementation of PIQLE, licensed under the GNU General Public License v3, is freely available at https://github.com/Bhattacharya-Lab/PIQLE .

rrQNet: Protein contact map quality estimation by deep evolutionary reconciliation.

Protein contact maps have proven to be a valuable tool in the deep learning revolution of protein structure prediction, ushering in the recent breakthrough by AlphaFold2. However, self-assessment of the quality of predicted structures are typically performed at the granularity of three-dimensional coordinates as opposed to directly exploiting the rotation- and translation-invariant two-dimensional (2D) contact maps. Here, we present rrQNet, a deep learning method for self-assessment in 2D by contact map quality estimation. Our approach is based on the intuition that for a contact map to be of high quality, the residue pairs predicted to be in contact should be mutually consistent with the evolutionary context of the protein. The deep neural network architecture of rrQNet implements this intuition by cascading two deep modules-one encoding the evolutionary context and the other performing evolutionary reconciliation. The penultimate stage of rrQNet estimates the quality scores at the interacting residue-pair level, which are then aggregated for estimating the quality of a contact map. This design choice offers versatility at varied resolutions from individual residue pairs to full-fledged contact maps. Trained on multiple complementary sources of contact predictors, rrQNet facilitates generalizability across various contact maps. By rigorously testing using publicly available datasets and comparing against several in-house baseline approaches, we show that rrQNet accurately reproduces the true quality score of a predicted contact map and successfully distinguishes between accurate and inaccurate contact maps predicted by a wide variety of contact predictors. The open-source rrQNet software package is freely available at https://github.com/Bhattacharya-Lab/rrQNet.

DisCovER: distance- and orientation-based covariational threading for weakly homologous proteins.

Threading a query protein sequence onto a library of weakly homologous structural templates remains challenging, even when sequence-based predicted contact or distance information is used. Contact-assisted or distance-assisted threading methods utilize only the spatial proximity of the interacting residue pairs for template selection and alignment, ignoring their orientation. Moreover, existing threading methods fail to consider the neighborhood effect induced by the query-template alignment. We present a new distance- and orientation-based covariational threading method called DisCovER by effectively integrating information from inter-residue distance and orientation along with the topological network neighborhood of a query-template alignment. Our method first selects a subset of templates using standard profile-based threading coupled with topological network similarity terms to account for the neighborhood effect and subsequently performs distance- and orientation-based query-template alignment using an iterative double dynamic programming framework. Multiple large-scale benchmarking results on query proteins classified as weakly homologous from the continuous automated model evaluation experiment and from the current literature show that our method outperforms several existing state-of-the-art threading approaches, and that the integration of the neighborhood effect with the inter-residue distance and orientation information synergistically contributes to the improved performance of DisCovER. DisCovER is freely available at https://github.com/Bhattacharya-Lab/DisCovER.

Recent Advances in Protein Homology Detection Propelled by Inter-Residue Interaction Map Threading.

Sequence-based protein homology detection has emerged as one of the most sensitive and accurate approaches to protein structure prediction. Despite the success, homology detection remains very challenging for weakly homologous proteins with divergent evolutionary profile. Very recently, deep neural network architectures have shown promising progress in mining the coevolutionary signal encoded in multiple sequence alignments, leading to reasonably accurate estimation of inter-residue interaction maps, which serve as a rich source of additional information for improved homology detection. Here, we summarize the latest developments in protein homology detection driven by inter-residue interaction map threading. We highlight the emerging trends in distant-homology protein threading through the alignment of predicted interaction maps at various granularities ranging from binary contact maps to finer-grained distance and orientation maps as well as their combination. We also discuss some of the current limitations and possible future avenues to further enhance the sensitivity of protein homology detection.

DeepRefiner: high-accuracy protein structure refinement by deep network calibration.

The DeepRefiner webserver, freely available at http://watson.cse.eng.auburn.edu/DeepRefiner/, is an interactive and fully configurable online system for high-accuracy protein structure refinement. Fuelled by deep learning, DeepRefiner offers the ability to leverage cutting-edge deep neural network architectures which can be calibrated for on-demand selection of adventurous or conservative refinement modes targeted at degree or consistency of refinement. The method has been extensively tested in the Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments under the group name 'Bhattacharya-Server' and was officially ranked as the No. 2 refinement server in CASP13 (second only to 'Seok-server' and outperforming all other refinement servers) and No. 2 refinement server in CASP14 (second only to 'FEIG-S' and outperforming all other refinement servers including 'Seok-server'). The DeepRefiner web interface offers a number of convenient features, including (i) fully customizable refinement job submission and validation; (ii) automated job status update, tracking, and notifications; (ii) interactive and interpretable web-based results retrieval with quantitative and visual analysis and (iv) extensive help information on job submission and results interpretation via web-based tutorial and help tooltips.

Hybridized distance- and contact-based hierarchical structure modeling for folding soluble and membrane proteins.

Crystallography and NMR system (CNS) is currently a widely used method for fragment-free ab initio protein folding from inter-residue distance or contact maps. Despite its widespread use in protein structure prediction, CNS is a decade-old macromolecular structure determination system that was originally developed for solving macromolecular geometry from experimental restraints as opposed to predictive modeling driven by interaction map data. As such, the adaptation of the CNS experimental structure determination protocol for ab initio protein folding is intrinsically anomalous that may undermine the folding accuracy of computational protein structure prediction. In this paper, we propose a new CNS-free hierarchical structure modeling method called DConStruct for folding both soluble and membrane proteins driven by distance and contact information. Rigorous experimental validation shows that DConStruct attains much better reconstruction accuracy than CNS when tested with the same input contact map at varying contact thresholds. The hierarchical modeling with iterative self-correction employed in DConStruct scales at a much higher degree of folding accuracy than CNS with the increase in contact thresholds, ultimately approaching near-optimal reconstruction accuracy at higher-thresholded contact maps. The folding accuracy of DConStruct can be further improved by exploiting distance-based hybrid interaction maps at tri-level thresholding, as demonstrated by the better performance of our method in folding free modeling targets from the 12th and 13th rounds of the Critical Assessment of techniques for protein Structure Prediction (CASP) experiments compared to popular CNS- and fragment-based approaches and energy-minimization protocols, some of which even using much finer-grained distance maps than ours. Additional large-scale benchmarking shows that DConStruct can significantly improve the folding accuracy of membrane proteins compared to a CNS-based approach. These results collectively demonstrate the feasibility of greatly improving the accuracy of ab initio protein folding by optimally exploiting the information encoded in inter-residue interaction maps beyond what is possible by CNS.

PolyFold: An interactive visual simulator for distance-based protein folding.

Recent advances in distance-based protein folding have led to a paradigm shift in protein structure prediction. Through sufficiently precise estimation of the inter-residue distance matrix for a protein sequence, it is now feasible to predict the correct folds for new proteins much more accurately than ever before. Despite the exciting progress, a dedicated visualization system that can dynamically capture the distance-based folding process is still lacking. Most molecular visualizers typically provide only a static view of a folded protein conformation, but do not capture the folding process. Even among the selected few graphical interfaces that do adopt a dynamic perspective, none of them are distance-based. Here we present PolyFold, an interactive visual simulator for dynamically capturing the distance-based protein folding process through real-time rendering of a distance matrix and its compatible spatial conformation as it folds in an intuitive and easy-to-use interface. PolyFold integrates highly convergent stochastic optimization algorithms with on-demand customizations and interactive manipulations to maximally satisfy the geometric constraints imposed by a distance matrix. PolyFold is capable of simulating the complex process of protein folding even on modest personal computers, thus making it accessible to the general public for fostering citizen science. Open source code of PolyFold is freely available for download at https://github.com/Bhattacharya-Lab/PolyFold. It is implemented in cross-platform Java and binary executables are available for macOS, Linux, and Windows.

QDeep: distance-based protein model quality estimation by residue-level ensemble error classifications using stacked deep residual neural networks.

MOTIVATION: Protein model quality estimation, in many ways, informs protein structure prediction. Despite their tight coupling, existing model quality estimation methods do not leverage inter-residue distance information or the latest technological breakthrough in deep learning that has recently revolutionized protein structure prediction. RESULTS: We present a new distance-based single-model quality estimation method called QDeep by harnessing the power of stacked deep residual neural networks (ResNets). Our method first employs stacked deep ResNets to perform residue-level ensemble error classifications at multiple predefined error thresholds, and then combines the predictions from the individual error classifiers for estimating the quality of a protein structural model. Experimental results show that our method consistently outperforms existing state-of-the-art methods including ProQ2, ProQ3, ProQ3D, ProQ4, 3DCNN, MESHI, and VoroMQA in multiple independent test datasets across a wide-range of accuracy measures; and that predicted distance information significantly contributes to the improved performance of QDeep. AVAILABILITY AND IMPLEMENTATION: https://github.com/Bhattacharya-Lab/QDeep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SPECS: Integration of side-chain orientation and global distance-based measures for improved evaluation of protein structural models.

Significant advancements in the field of protein structure prediction have necessitated the need for objective and robust evaluation of protein structural models by comparing predicted models against the experimentally determined native structures to quantitate their structural similarities. Existing protein model versus native similarity metrics either consider the distances between alpha carbon (Cα) or side-chain atoms for computing the similarity. However, side-chain orientation of a protein plays a critical role in defining its conformation at the atomic-level. Despite its importance, inclusion of side-chain orientation in structural similarity evaluation has not yet been addressed. Here, we present SPECS, a side-chain-orientation-included protein model-native similarity metric for improved evaluation of protein structural models. SPECS combines side-chain orientation and global distance based measures in an integrated framework using the united-residue model of polypeptide conformation for computing model-native similarity. Experimental results demonstrate that SPECS is a reliable measure for evaluating structural similarity at the global level including and beyond the accuracy of Cα positioning. Moreover, SPECS delivers superior performance in capturing local quality aspect compared to popular global Cα positioning-based metrics ranging from models at near-experimental accuracies to models with correct overall folds-making it a robust measure suitable for both high- and moderate-resolution models. Finally, SPECS is sensitive to minute variations in side-chain χ angles even for models with perfect Cα trace, revealing the power of including side-chain orientation. Collectively, SPECS is a versatile evaluation metric covering a wide spectrum of protein modeling scenarios and simultaneously captures complementary aspects of structural similarities at multiple levels of granularities. SPECS is freely available at http://watson.cse.eng.auburn.edu/SPECS/.

Evaluating the significance of contact maps in low-homology protein modeling using contact-assisted threading.

The development of improved threading algorithms for remote homology modeling is a critical step forward in template-based protein structure prediction. We have recently demonstrated the utility of contact information to boost protein threading by developing a new contact-assisted threading method. However, the nature and extent to which the quality of a predicted contact map impacts the performance of contact-assisted threading remains elusive. Here, we systematically analyze and explore this interdependence by employing our newly-developed contact-assisted threading method over a large-scale benchmark dataset using predicted contact maps from four complementary methods including direct coupling analysis (mfDCA), sparse inverse covariance estimation (PSICOV), classical neural network-based meta approach (MetaPSICOV), and state-of-the-art ultra-deep learning model (RaptorX). Experimental results demonstrate that contact-assisted threading using high-quality contacts having the Matthews Correlation Coefficient (MCC) ≥ 0.5 improves threading performance in nearly 30% cases, while low-quality contacts with MCC <0.35 degrades the performance for 50% cases. This holds true even in CASP13 dataset, where threading using high-quality contacts (MCC ≥ 0.5) significantly improves the performance of 22 instances out of 29. Collectively, our study uncovers the mutual association between the quality of predicted contacts and its possible utility in boosting threading performance for improving low-homology protein modeling.

Does inclusion of residue-residue contact information boost protein threading?

Template-based modeling is considered as one of the most successful approaches for protein structure prediction. However, reliably and accurately selecting optimal template proteins from a library of known protein structures having similar folds as the target protein and making correct alignments between the target sequence and the template structures, a template-based modeling technique known as threading, remains challenging, particularly for non- or distantly-homologous protein targets. With the recent advancement in protein residue-residue contact map prediction powered by sequence co-evolution and machine learning, here we systematically analyze the effect of inclusion of residue-residue contact information in improving the accuracy and reliability of protein threading. We develop a new threading algorithm by incorporating various sequential and structural features, and subsequently integrate residue-residue contact information as an additional scoring term for threading template selection. We show that the inclusion of contact information attains statistically significantly better threading performance compared to a baseline threading algorithm that does not utilize contact information when everything else remains the same. Experimental results demonstrate that our contact based threading approach outperforms popular threading method MUSTER, contact-assisted ab initio folding method CONFOLD2, and recent state-of-the-art contact-assisted protein threading methods EigenTHREADER and map_align on several benchmarks. Our study illustrates that the inclusion of contact maps is a promising avenue in protein threading to ultimately help to improve the accuracy of protein structure prediction.

refineD: improved protein structure refinement using machine learning based restrained relaxation.

MOTIVATION: Protein structure refinement aims to bring moderately accurate template-based protein models closer to the native state through conformational sampling. However, guiding the sampling towards the native state by effectively using restraints remains a major issue in structure refinement. RESULTS: Here, we develop a machine learning based restrained relaxation protocol that uses deep discriminative learning based binary classifiers to predict multi-resolution probabilistic restraints from the starting structure and subsequently converts these restraints to be integrated into Rosetta all-atom energy function as additional scoring terms during structure refinement. We use four restraint resolutions as adopted in GDT-HA (0.5, 1, 2 and 4 Å), centered on the Cα atom of each residue that are predicted by ensemble of four deep discriminative classifiers trained using combinations of sequence and structure-derived features as well as several energy terms from Rosetta centroid scoring function. The proposed method, refineD, has been found to produce consistent and substantial structural refinement through the use of cumulative and non-cumulative restraints on 150 benchmarking targets. refineD outperforms unrestrained relaxation strategy or relaxation that is restrained to starting structures using the FastRelax application of Rosetta or atomic-level energy minimization based ModRefiner method as well as molecular dynamics (MD) simulation based FG-MD protocol. Furthermore, by adjusting restraint resolutions, the method addresses the tradeoff that exists between degree and consistency of refinement. These results demonstrate a promising new avenue for improving accuracy of template-based protein models by effectively guiding conformational sampling during structure refinement through the use of machine learning based restraints. AVAILABILITY AND IMPLEMENTATION: http://watson.cse.eng.auburn.edu/refineD/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.