DeepFRET, a software for rapid and automated single-molecule FRET data classification using deep learning.

ABSTRACT

Single-molecule Förster Resonance energy transfer (smFRET) is an adaptable method for studying the structure and dynamics of biomolecules. The development of high throughput methodologies and the growth of commercial instrumentation have outpaced the development of rapid, standardized, and automated methodologies to objectively analyze the wealth of produced data. Here we present DeepFRET, an automated, open-source standalone solution based on deep learning, where the only crucial human intervention in transiting from raw microscope images to histograms of biomolecule behavior, is a user-adjustable quality threshold. Integrating standard features of smFRET analysis, DeepFRET consequently outputs the common kinetic information metrics. Its classification accuracy on ground truth data reached >95% outperforming human operators and commonly used threshold, only requiring ~1% of the time. Its precise and rapid operation on real data demonstrates DeepFRET's capacity to objectively quantify biomolecular dynamics and the potential to contribute to benchmarking smFRET for dynamic structural biology.

Proteins are folded into particular shapes in order to carry out their roles in the cell. However, their structures are not rigid proteins bend and rotate in response to their environment. Identifying these movements is an important part of understanding how proteins work and interact with each other. Unfortunately, when researchers study the structures of proteins, they often look at the 'average' shape a protein takes, missing out on other conformations the protein might only be in temporarily. An important technique for studying protein flexibility is known as single molecule Förster resonance energy transfer (FRET). In this technique, two light-sensitive tags are attached to the same protein molecule and give off a signal when they come into close contact. This nano-scale sensor allows structural biologists to get information from individual protein movements that can be lost when looking at the average conformations of proteins. Advances in the instruments used to perform FRET have made observing the motion of individual proteins more widely accessible to non-specialists, but the analysis of the data that these instruments produce still requires a high level of expertise. To lower the barrier for non-specialists to use the technology, and to ensure that experiments can be reproduced on different instruments and by different researchers, Thomsen et al. have developed a new way to automate the data analysis. They used machine learning technology to recognize, filter and characterize data so as to produce reliable results, with the user only needing to perform a couple of steps. This new analysis approach could help expand the use of single-molecule FRET to different fields , allowing researchers to investigate the importance of protein flexibility for certain diseases, or to better understand the roles that proteins have in a cell.