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Attaching fluorescent dyes to biomolecules is essential for assays in biology, biochemistry, biophysics, biomedicine and imaging. A systematic approach for the selection of suitable labeling sites in macromolecules, particularly proteins, is missing. We present a quantitative strategy to identify such protein residues using a naïve Bayes classifier. Analysis of >100 proteins with ~400 successfully labeled residues allows to identify four parameters, which can rank residues via a single metric (the label score). The approach is tested and benchmarked by inspection of literature data and experiments on the expression level, degree of labelling, and success in FRET assays of different bacterial substrate binding proteins. With the paper, we provide a python package and webserver ( https://labelizer.bio.lmu.de/ ), that performs an analysis of a pdb-structure (or model), label score calculation, and FRET assay scoring. The approach can facilitate to build up a central open-access database to continuously refine the label-site selection in proteins. Gebhardt and colleagues developed a computational method using a naïve Bayes classifier to identify optimal protein labelling sites. Their analysis of 100+ proteins revealed four predictive parameters, leading to a Python package and a web-tool for protein structure analysis and labelling score calculations.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology. An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.

An essential requirement for the use of fluorescent dyes in biomedicine, molecular biology, biochemistry, biophysics and optical imaging is their (covalent) attachment to biomolecules. There is, however, no systematic and automated approach for the selection of suitable labeling sites in macromolecules, which is particular problematic for proteins. Here, we present a general and quantitative strategy to identify optimal residues for protein labeling using a naïve Bayes classifier. Based on a literature search and bioinformatics analysis of >100 proteins with ~400 successfully labeled residues, we identified four parameters, which we combined into a labeling score to rank residues for their suitability as a label-site. The utility of our approach for the systematic selection of single residues and of residue pairs for FRET experiments is supported by data from the literature and by new experiments on different proteins. To make the method available to a large community of researchers, we developed a python package called “labelizer”, that performs an analysis of a pdb-structure (or structural models), label score calculation, and FRET assay scoring. We further provide a webserver (https://labelizer.bio.lmu.de/) to conveniently apply our approach and to build up a central open-access database of (non-)successfully labeled protein residues to continuously improve and refine the labelizer approach.

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.Competing Interest StatementThe authors have declared no competing interest.

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than 0.06, corresponding to an interdye distance precision of less than 0.2 nm and accuracy of less than 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to less than 10% of the Foerster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology. Competing Interest Statement Tim Craggs and Achilles Kapanidis, two of the authors are founders of different companies selling single-molecule fluorescence microscopes (Exciting Instruments, Oxford Nanoimager).

Mucus consistency affects voice physiology and is connected to voice disorders. Nevertheless, the rheological characteristics of human laryngeal mucus from the vocal folds remain unknown. Knowledge about mucus viscoelasticity enables fabrication of artificial mucus with natural properties, more realistic ex-vivo experiments and promotes a better understanding and improved treatment of dysphonia with regard to mucus consistency. We studied human laryngeal mucus samples from the vocal folds with two complementary approaches: 19 samples were successfully applied to particle tracking microrheology (PTM) and five additional samples to oscillatory shear rheology (OSR). Mucus was collected by experienced laryngologists from patients together with demographic data. The analysis of the viscoelasticity revealed diversity among the investigated mucus samples according to their rigidity (absolute G′ and G″). Moreover some samples revealed throughout solid-like character (G′ > G″), whereas some underwent a change from solid-like to liquid-like (G′ < G″). This led to a subdivision into three groups. We assume that the reason for the differences is a variation in the hydration level of the mucus, which affects the mucin concentration and network formation factors of the mucin mesh. The demographic data could not be correlated to the differences, except for the smoking behavior. Mucus of predominant liquid-like character was associated with current smokers.

Foot problems are a common concern in elephant husbandry. Studies on this topic with sample sizes greater than 100 animals have only been carried out in North America. We investigated foot health of 243 Asian elephants (Elephas maximus) in 69 European institutions. During on-site visits between August 2016 and July 2017, standardized pictures were taken of each elephant's nails and pads. The pictures were analyzed with respect to pathological lesions (i.e. nail cracks, abscesses), care issues (i.e. minor abnormalities, which are easily resolvable with routine foot work), and pad structure. Of all analyzed nails and pads, 35.6% revealed varying degrees of pathological lesions, with minor nail cracks and overgrown cuticles with attachment to the nails being most frequently observed. The most lateral nail (N5) on both front feet demonstrated the highest percentage of pathological lesions, providing support to a separate study showing that the mean peak pressure of an elephant's foot occurs along the most lateral digits; however, this was not observed along the most lateral nail (N5) of the rear feet. Three (of 243) elephants did not show any pathological lesions in their feet. The most common issues requiring foot care were fissures in the nail sole. The structure of the pads was categorized in four grades reflecting the percentage of surface marked by sulci. These four grades occurred at nearly equal frequency. Pearson product moment correlations revealed no significant association between the frequency of care issues and pathological lesions per nail. Despite this finding, it may be prudent to implement husbandry protocols that could alleviate commonly observed pathological and care foot issues in captive Asian elephants. A standardized approach to evaluate elephant foot health will provide a more objective way to monitor responses to management and medical decisions and ultimately contribute to the overall wellbeing of elephants in human care.