FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Bestandighetsstudie av Ultra-High Performance Concrete (UHPC) : En forskning om påvirkningen av rissvidde, overdekning og eksponeringsmiljø med hensyn på betongens beskyttelsesevne mot armeringskorrosjon

Master's thesis in Civil and structural engineering (BYG508)Ultra-High Performance Concrete (UHPC) was developed in research to increase the strength and durability of concrete. The material has shown remarkable ductility, durabilityand strength properties in laboratory tests, but has not beenmuch tested in practice. This master thesis presents an experiment to investigate the durability of UHPC in two marine environments in view of the risk of rebar corrosion. Factors under consideration are cover depth and crack width to observe how this will influence the penetration of aggressive substances that can cause corrosion. To investigate the corrosion state and chloride content, electrochemical impedance spectroscopy (EIS) and Volhard ́s method were performed. This was done in orderto observe the permeability of UHPC.The results showthat the EIS measurements for the UHPC beams have significantly greater corrosion resistance compared to thestandard concretebeams,SV-40. UHPC has a resistance of almost tenfold of the SV-40. The result from the EIS-measurement for the corrosive rebar, makes a too weak comparison basis to draw a conclusion onwhether there is corrosion in the different exposure environment. Disregarding that there iscorrosion on the rebars, the UHPC beams could be dimensioned with a smaller concrete cover and a larger crack width compared with the requirements from the standards. Overall, this experiment shows that UHPC has a superior corrosion resistance in practice compared to SV-40

Bestandighetsstudie av Ultra-High Performance Concrete (UHPC) : En forskning om påvirkningen av rissvidde, overdekning og eksponeringsmiljø med hensyn på betongens beskyttelsesevne mot armeringskorrosjon

Ultra-High Performance Concrete (UHPC) was developed in research to increase the strength and durability of concrete. The material has shown remarkable ductility, durabilityand strength properties in laboratory tests, but has not beenmuch tested in practice. This master thesis presents an experiment to investigate the durability of UHPC in two marine environments in view of the risk of rebar corrosion. Factors under consideration are cover depth and crack width to observe how this will influence the penetration of aggressive substances that can cause corrosion. To investigate the corrosion state and chloride content, electrochemical impedance spectroscopy (EIS) and Volhard ́s method were performed. This was done in orderto observe the permeability of UHPC.The results showthat the EIS measurements for the UHPC beams have significantly greater corrosion resistance compared to thestandard concretebeams,SV-40. UHPC has a resistance of almost tenfold of the SV-40. The result from the EIS-measurement for the corrosive rebar, makes a too weak comparison basis to draw a conclusion onwhether there is corrosion in the different exposure environment. Disregarding that there iscorrosion on the rebars, the UHPC beams could be dimensioned with a smaller concrete cover and a larger crack width compared with the requirements from the standards. Overall, this experiment shows that UHPC has a superior corrosion resistance in practice compared to SV-40