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We introduce software for reading, writing and processing fluorescence single-molecule and image spectroscopy data and developing analysis pipelines that unifies various spectroscopic analysis tools. Our software can be used for processing multiple experiment types, e.g., for time-resolved single-molecule (sm) spectroscopy, laser scanning microscopy, fluorescence correlation spectroscopy, and image correlation spectroscopy. The software is file format agnostic, processes and outputs multiple time-resolved data formats. Thereby our software eliminates the need for data conversion and mitigates data archiving issues.

Single-molecule F\"orster Resonance Energy Transfer (smFRET) is ideally suited to resolve the dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for the quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with solely static molecules in similar conformational states. To address these problems, we developed an integrative analysis framework that combines the information from FRET-lines, time-correlated single photon counting, and fluorescence correlation spectroscopy. While individually, these methodologies provide ambiguous results for quantitatively characterize the dynamics in complex kinetic networks, the global analysis enables accurate determination of the number of states, their kinetic connectivity, and the transition rate constants. To challenge the potential of smFRET to study multi-state kinetic networks, we benchmark the framework using synthetic data of three-state systems with different kinetic connectivity and interconversion rates., Comment: Main Text (31 pages, 9 figures, 3 tables) with Supporting Information (40 pages, 10 figures, 2 tables)

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule F\"orster Resonance Energy Transfer (smFRET) is a powerful approach to inform on the structure and the dynamics of labeled molecules. If the dynamics occur on the sub-millisecond timescale, capturing and quantifying conformational dynamics can be challenging by intensity-based smFRET. Multiparameter fluorescence detection (MFD) addresses this challenge by simultaneously registering intensities and fluorescence lifetimes. Together, the mean donor fluorescence lifetime and the fluorescence intensities inform on the variance, and the mean FRET efficiency tells the conformational dynamics. Here, we present a general framework that relates average fluorescence lifetimes and intensities in smFRET counting histograms. Using this framework, we show how to compute parametric relations (FRET-lines) of these observables that facilitate a graphical interpretation of experimental data, can be used to test models, identify conformational states, resolve the connectivity of states, and can be applied to unstructured systems to infer properties of polymer chains or study fast protein folding. To simplify the graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data and show how to decouple the motion of the fluorescence labels from the conformational dynamics of the biomolecule., Comment: Main Text (47 pages, 14 figures, 3 tables) with Supporting Information (22 pages, 2 figures)

Single-molecule F\"{o}rster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins with structured domains that undergo a conformational transition for GTP-controlled oligomerization to exert their function as part of the innate immune system of mammalian cells - attacking intra-cellular parasites by disrupting their membranes. The structural basis and mechanism of this process is unknown. Therefore, we apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the $\mu$s-regime and structurally characterize conformers being essential that hGBP1 can open like a pocketknife for oligomerization. This unveils the intrinsic flexibility, a GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis as functional design principles of hGBP1 that control its reversible oligomerization in polar assemblies and the subsequent formation of condensates., Comment: 83 pages

FRET experiments can yield state-specific structural information on complex dynamic biomolecular assemblies. However, FRET experiments need to be combined with computer simulations to overcome their sparsity. We introduce (i) an automated FRET experiment design tool determining optimal FRET pairs for structural modeling, (ii) a protocol for efficient FRET-assisted computational structural modeling at multiple scales, and (iii) a quantitative quality estimate for judging the accuracy of determined structures. We tested against simulated and experimental data., Comment: 18 pages, 2 figures, 1 table

We used a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action. By unraveling the kinetic and dynamic interplay of the conformational states, we sought to elucidate the dynamic structural biology of T4L. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, we characterized three short-lived conformational states within the conformational landscape of the T4L over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen known T4L structures, revealed that T4L in solution mainly adopts the known open and closed states in exchange at 4 $\mu$s. A newly found minor state, undisclosed by at present more than 500 crystal structures of T4L and sampled at 230 $\mu$s, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit is anticipated to accelerate the development of dynamic structural biology., Comment: 43 pages, 7 figures, Supporting Information 50 pages