Cold Spot SCANNER: Colab Notebook for predicting cold spots in protein–protein interfaces

Sagara N.S. Gurusinghe, Julia M. Shifman

Research output: Contribution to journalArticlepeer-review


Background: Protein–protein interactions (PPIs) are conveyed through binding interfaces or surface patches on proteins that become buried upon binding. Structural and biophysical analysis of many protein–protein interfaces revealed certain unique features of these surfaces that determine the energetics of interactions and play a critical role in protein evolution. One of the significant aspects of binding interfaces is the presence of binding hot spots, where mutations are highly deleterious for binding. Conversely, binding cold spots are positions occupied by suboptimal amino acids and several mutations in such positions could lead to affinity enhancement. While there are many software programs for identification of hot spot positions, there is currently a lack of software for cold spot detection. Results: In this paper, we present Cold Spot SCANNER, a Colab Notebook, which scans a PPI binding interface and identifies cold spots resulting from cavities, unfavorable charge-charge, and unfavorable charge-hydrophobic interactions. The software offers a Py3DMOL-based interface that allows users to visualize cold spots in the context of the protein structure and generates a zip file containing the results for easy download. Conclusions: Cold spot identification is of great importance to protein engineering studies and provides a useful insight into protein evolution. Cold Spot SCANNER is open to all users without login requirements and can be accessible at:

Original languageAmerican English
Article number172
JournalBMC Bioinformatics
Issue number1
StatePublished - Dec 2024


  • Binding affinity
  • Binding interface
  • Cavities
  • Cold spots
  • Protein–protein interactions

All Science Journal Classification (ASJC) codes

  • Applied Mathematics
  • Molecular Biology
  • Structural Biology
  • Biochemistry
  • Computer Science Applications


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