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# Appendix 1

## Simulation software

* simFCS (https://www.lfd.uci.edu/globals/) was developed for simulating various modalities in fluorescence, beginning with FCS. Using two channels, the simulation of burst analysis smFRET data can be performed. The concentration, diffusion coefficient and molecular brightness in different channels and dynamics between them can be adjusted.

* PyBroMo (https://github.com/tritemio/PyBroMo) is a Python-based software package for simulating free-diffusion single-molecule fluorescence detection, including differences in translational diffusion coefficients, in concentrations, or in dye brightnesses and exchange dynamics between conformational states (currently between two states). This approach has been employed in a few recent works (Ingargiola et al., 2017; Lerner et al., 2018b; Hagai and Lerner, 2019).

* PAM software package (https://www.cup.uni-muenchen.de/pc/lamb/software/pam.html). The PAM software package is a MATLAB-based software that can simulate smFRET experiments of freely diffusing or immobilized molecules, including fluorescence lifetime and anisotropy as well as exchange dynamics between multiple conformational states (up to 8) Szalai et al., 2021 Schrimpf et al., 2018).

* The Burbulator software within the MFD program package, programmed in LabVIEW (Dimura et al., 2016; Felekyan et al., 2012; Kalinin et al., 2010b) (https://www.mpc.hhu.de/software/3-software-package-for-mfd-fcs-and-mfis), can simulate smFRET experiments (with and without diffusion) combined fluorescence lifetime and anisotropy as well as exchange dynamics between multiple conformational states (up to 8). These tools have been applied to benchmark novel quantitative analysis methods to obtain structural and kinetic information.

* Fretica (https://schuler.bioc.uzh.ch/programs/) enables the simulation of single-molecule multi-channel-detection of immobilized molecules and mixtures of freely diffusing species, including a dynamic exchange between an arbitrary number of conformational states (König et al., 2015; Zosel et al., 2018).

* The MATLAB-based MASH-FRET software package (Börner et al., 2018) (https://rna-fretools.github.io/MASH-FRET/) has been applied to evaluate transition detection and state identification algorithms used in particular for time-binned smFRET trajectories (Hadzic et al., 2018) as well as for spot detection in single-molecule videos (SMV).

* The python-based software DeepFRET (Thomsen et al., 2020) comes with a trace simulator capable of simulating traces with 17 adjustable features that include the number of FRET states, their values, noise level, transition probability and more (https://github.com/hatzakislab/DeepFRET-GUI).