Improvement of protein tertiary and quaternary structure predictions using the ReFOLD4 refinement method and the AlphaFold2 recycling process

Abstract

MotivationThe accuracy gap between predicted and experimental structures has been significantly reduced following the development of AlphaFold2. However, for further studies, such as drug discovery and protein design, AlphaFold2 structures need to be representative of proteins in solution, yet AlphaFold2 was trained to generate only a few structural conformations rather than a conformational landscape. In previous CASP experiments, MD simulation-based methods have been widely used to improve the accuracy of single 3D models. However, these methods are highly computationally intensive and less applicable for practical use in large-scale applications. Despite this, the refinement concept can still provide a better understanding of conformational dynamics and improve the quality of 3D models at a modest computational cost. Here, our ReFOLD4 pipeline was adopted to provide the conformational landscape of AlphaFold2 predictions while maintaining high model accuracy. In addition, the AlphaFold2 recycling process was utilised to improve 3D models by using them as custom template inputs for tertiary and quaternary structure predictions. ResultsAccording to the Molprobity score, 94% of the generated 3D models by ReFOLD4 were improved. As measured by average change in lDDT, AlphaFold2 recycling showed an improvement rate of 87.5% (using MSAs) and 81.25% (using single sequences) for monomeric AF2 models and 100% (MSA) and 97.8% (single sequence) for monomeric non-AF2 models. By the same measure, the recycling of multimeric models showed an improvement rate of as much as 80% for AF2 models and 94% for non-AF2 models. The AlphaFold2 recycling processes and ReFOLD4 method can be combined very efficiently to provide conformational landscapes at the AlphaFold2-accuracy level, while also significantly improving the global quality of 3D models for both tertiary and quaternary structures, with much less computational complexity than traditional refinement methods.