Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Stefan Niekamp, Jongmin Sung, Walter Huynh, Gira Bhabha, Ronald D. Vale, Nico Stuurman
2019 Proceedings of the National Academy of Sciences of the United States of America  
Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about
more » ... n and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.
doi:10.1073/pnas.1815826116 pmid:30770448 pmcid:PMC6410877 fatcat:g75onbx5xrh5vfppkweih5undi