AbstractMotivationWith the development of sequencing techniques, the discovery of new proteins significantly exceeds the human capacity and resources for experimentally characterizing protein functions. Localization, EC numbers, and GO terms with the structure-based Cutoff Scanning Matrix (LEGO-CSM) is a comprehensive web-based resource that fills this gap by leveraging the well-established and robust graph-based signatures to supervised learning models using both protein sequence and structure information to accurately model protein function in terms of Subcellular Localization, Enzyme Commission (EC) numbers, and Gene Ontology (GO) terms.ResultsWe show our models perform as well as or better than alternative approaches, achieving area under the receiver operating characteristic curve of up to 0.93 for subcellular localization, up to 0.93 for EC, and up to 0.81 for GO terms on independent blind tests.Availability and implementationLEGO-CSM’s web server is freely available athttps://biosig.lab.uq.edu.au/lego_csm. In addition, all datasets used to train and test LEGO-CSM’s models can be downloaded athttps://biosig.lab.uq.edu.au/lego_csm/data.