This framework provides a clearer understanding of how methodological shifts have shaped the capabilities, limitations, and practical roles of recent models.
Abstract
Accurate protein structure prediction is fundamental to structural biology because protein structure underlies molecular function and provides a basis for mechanistic interpretation. Recent advances in deep learning have transformed the field from multiple sequence alignment (MSA)-driven monomer folding into broader frameworks capable of modeling protein complexes and increasingly heterogeneous molecular systems. Existing reviews have summarized this progress from the perspectives of representative models, application domains, and protein design. Building on these efforts, this review focuses on the methodological evolution of the field itself. It examines recent developments through three closely related dimensions: representations and data, architectures and learning strategies, and confidence and evaluation. Within this perspective, the field is organized into four methodological phases and three cross-cutting transitions: from explicit evolutionary coupling features and early contact prediction to learned sequence representations in AlphaFold2, RoseTTAFold, and ESMFold; from protein-only monomer folding to increasingly integrated modeling of heterogeneous molecular systems in AlphaFold-Multimer, RoseTTAFoldNA, and AlphaFold3; and, more recently, from prediction-oriented structure inference to design-oriented generative modeling in RFdiffusion and related frameworks. This framework provides a clearer understanding of how methodological shifts have shaped the capabilities, limitations, and practical roles of recent models.
This mini review traces the evolution of AI-driven methods in protein research, from early residue-contact prediction using coevolutionary information to transformative breakthroughs, the rise of protein language models (PLMs), and the emerging era of generative design and functional modeling.
Guodong Min, Huan Peng· Methods in molecular biology· 0 citations
It is concluded that molecular dynamics has an important place in improving the physicality of existing protein structure prediction paradigms, leading to the development of the Subspace Relaxation Operator (SRO).
Colin Baker, Pranav Mahableshwarkar, Ritambhara Singh et al.· 0 citations
Recent AI advances have enabled protein structure prediction at near-experimental accuracy, largely solving the problem of identifying a dominant conformation from sequence. Many proteins, however, function as dynamic systems populating multiple conformational states with activity emerging from shifts in relative occupancy--an incomplete picture when reduced to one structure. Here, we argue that structure prediction should be reformulated as a state-space inference problem: recovering not one conformation's coordinates but accessible states, their energetic and kinetic relationships, context dependence, and responses to perturbations. We review emerging strategies--deep learning ensemble generators, physics-based simulations, and experimental constraints--and outline a roadmap toward state-space prediction.
Devlina Chakravarty, Justin J. Miller, Da Teng et al.· 0 citations
Recent advances in de novo protein design have enabled the generation of diverse novel proteins. However, a fundamental challenge remains: even when an amino acid sequence is designed with the target structure as the most stable conformation, there is currently no reliable computational method for assessing whether the target structure is sufficiently stabilized relative to alternative conformations. While experimental realization of the intended fold requires the target structure to be thermodynamically favored by a large free‐energy gap, the absence of a quantitative measure of folding stability makes it difficult to distinguish reliable from unreliable designs. Here, we propose the Residue‐attributed Interpretable Neural network for predicting Absolute folding free energy by Merging structure and sequence Information (RINAMI), a machine learning model that predicts the absolute folding free energy (ΔG) of proteins from their three‐dimensional structures and amino acid sequences. RINAMI integrates structure‐ and sequence‐based representations derived from ProteinMPNN and Evolutionary Scale Modeling 2 (ESM2) using a multi‐head cross‐attention mechanism that contextualizes sequence‐derived signals within the structural environment. Benchmarking RINAMI on both natural and designed proteins from the Mega‐scale and Maxwell datasets shows that it outperforms the tested existing approaches, achieving higher correlations with experimental measurements and improved or comparable prediction errors. An ablation study supports the contribution of sequence–structure integration for predictive accuracy. In addition, RINAMI exhibits strong interpretability by capturing key physicochemical effects, including the destabilizing effect of buried hydrophilic residues, the stabilizing effect of buried hydrophobic residues, and the characteristics of cysteine. Together, these results establish RINAMI as an accurate and interpretable framework for ΔG prediction and provide a practical computational tool for evaluating and prioritizing protein designs prior to experimental testing.
Naoki Tomita, G. Chikenji· Protein Science· 0 citations
Multiple sequence alignment (MSA) Pairformer is presented, a protein language model that builds on AlphaFold2/3's bidirectional refinement between sequence and pairwise residue representations to accurately model the evolution of protein-protein interactions, despite training exclusively on individual chains.
Yo Akiyama, Zhidian Zhang, Olivia Tang et al.· Cell· 2 citations
Proteins are critical biomolecular machines that populate ensembles of interconverting conformations. Many biological processes depend on transitions between metastable states. Although molecular dynamics (MD) simulations provide a physically grounded route to characterize these motions, routine sampling of large-scale conformational transitions remains computationally demanding. Recent advances in protein structure prediction have created new opportunities for ensemble generation, but many existing approaches require noising inputs, task-specific training, supervised fitting on extensive MD data, or experimentally-informed restraints. Here, we introduce Pi-Ensemble (Predicting Interpolated Ensemble), a sequence-guided framework for generating protein conformational ensembles interpolating between two structural anchor states. Unlike previous methods, Pi-Ensemble alternately leverages inverse-folding and structure-prediction models to propose intermediate conformations between known protein states, generating diverse ensembles without additional training. We evaluate Pi-Ensemble across diverse protein systems, including enzymes, transporters, receptors, and benchmark cases with reference MD simulations or experimental Double Electron-Electron Resonance (DEER) data. Pi-Ensemble recovers physically plausible intermediate conformations, captures transition pathways observed in large-scale MD simulations, and generates structures consistent with experimental distance distributions. Furthermore, Pi-Ensemble-generated conformations provide effective starting seeds for parallel MD simulations, improving conformational exploration and accelerating convergence relative to simulations initiated only from endpoint structures. These results establish sequence-guided structural interpolation as a practical strategy for probing protein conformational landscapes. By generating diverse and physically reasonable conformational proposals without long-timescale MD or model retraining, Pi-Ensemble provides an extensible framework for studying protein flexibility, guiding adaptive sampling, and accelerating mechanistic investigations of protein function.
Hassan Nadeem, D. Kleiman, Yuming Zhou et al.· bioRxiv· 0 citations