ProtASR is an evolutionary framework to reconstruct ancestral protein sequences accounting for structural constraints. It is known that protein evolution is influenced by the protein structure. However, most of current phylogenetic frameworks do not implement structurally constrained substitution models of evolution due to their mathematical complexity. We have recently developed a set of structurally constrained substitution (SCS) models of protein evolution that consider positive and negative design and that can generate site-specific evolutionary parameters for a likelihood function. We already found that SCS models can better fit real protein evolution with respect to the traditional empirical substitution models in terms of maximum likelihood (AIC criterion) and amino acid distribution across sites. ProtASR applies these SCS models to perform a fast and accurate inference of ancestral protein sequences, hence accounting for structural constrains. ProtASR, through the implemented SCS models, can generate ancestral proteins that are more realistic than proteins generated with empirical substitution models and other SCS models. The package ProtASR2.2.zip includes source code and executable files, detailed documentation, several practical examples with both input and output files. ProtASR is freely available and open-source. References Last version (ProtASR 2.2): Arenas M & Bastolla U. 2019. ProtASR2: Ancestral Reconstruction of Protein Sequences accounting for Folding Stability. Methods in Ecology and Evolution. In press. Help Do not hesitate to contact us (miguelmmmab@gmail.com) for any question.

Acknowledgments This work was supported by the Spanish Ministery of Economy and Competitivity [RYC-2015-18241 to M.A., BIO2016-79043-P to U.B.]. MA was also funded by the Xunta de Galicia "ED431F 2018/08". Research at the CBMSO is facilitated by the Fundación Ramón Areces.

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