Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.

Changes in protein sequence can have dramatic effects on how proteins fold, their stability and dynamics. Over the last 20 years, pioneering methods have been developed to try to estimate the effects of missense mutations on protein stability, leveraging growing availability of protein 3D structures. These, however, have been developed and validated using experimentally derived structures and biophysical measurements. A large proportion of protein structures remain to be experimentally elucidated and, while many studies have based their conclusions on predictions made using homology models, there has been no systematic evaluation of the reliability of these tools in the absence of experimental structural data. We have, therefore, systematically investigated the performance and robustness of ten widely used structural methods when presented with homology models built using templates at a range of sequence identity levels (from 15% to 95%) and contrasted performance with sequence-based tools, as a baseline. We found there is indeed performance deterioration on homology models built using templates with sequence identity below 40%, where sequence-based tools might become preferable. This was most marked for mutations in solvent exposed residues and stabilizing mutations. As structure prediction tools improve, the reliability of these predictors is expected to follow, however we strongly suggest that these factors should be taken into consideration when interpreting results from structure-based predictors of mutation effects on protein stability.