scholarly journals High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level

2017 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Shape ◽  
High Accuracy ◽  
Distance Measurements ◽  
Distance Information ◽  
Wide Range

To uncover the mechanisms of molecular machines it is useful to probe their structural conformations. Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for measuring intra-molecular shape changes of single-molecules, but is confined to distances of 2-8 nm. Current super-resolution measurements are error prone at <25 nm. Thus, reliable high-throughput distance information between 8-25 nm is currently difficult to achieve. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1 nm accuracy at any distance >2 nm, using a standard TIRF microscope and open-source software. We applied our two-color localization method to uncover a ∼4 nm conformational change in the “stalk” of the motor protein dynein, revealing unexpected flexibility in this antiparallel coiled-coil domain. These new methods enable high-accuracy distance measurements of single-molecules that can be used over a wide range of length scales.

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

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Level ◽  
Wide Range ◽  
Different Color

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 localization 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.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

MATHEMATICAL TREATMENTS THAT SOLVE SINGLE MOLECULES

2013 ◽  
Vol 08 (03n04) ◽  
pp. 109-136 ◽  
Author(s):  
OPHIR FLOMENBOM
Keyword(s):  
Single Molecule ◽  
Likelihood Function ◽  
Single Molecules ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Real Data ◽  
Discrete Data ◽  
Special Issue ◽  
Discrete State ◽  
Accurate Model

In this article, we talk about the ways that scientists can solve single molecule trajectories. Solving single molecules, that is, finding the model from the data, is complicated at least as much as measuring single molecules. We must filter the noise and take care of every step in the analysis when constructing the most accurate model from the data. Here, we present valuable solutions. Ways that solve clean discrete data are first presented. We review here our reduced dimensions forms (RDFs): unique models that are canonical forms of discrete data, and the statistical and numerical toolbox that builds a RDF from finite, clean, two-state data. We then review our most recent filter that "tackles" the noise when measuring two state noisy photon trajectories. The filter is a numerical algorithm with various special statistical treatments that is based on a general likelihood function that we have developed recently. We show the strengths of the filter (also over other approaches) and talk about its various new variants. This filter (with minor adjustments) can solve the noise in any discrete state trajectories, yet, extensions are needed in "tackling" the noise from other data, e.g. continuous data. Only the combined procedures enable creating the most accurate model from noisy discrete trajectories from single molecules. These concepts and methods (with adjustments) are valuable also when solving continuous trajectories and fluorescence resonance energy transfer trajectories. We also present a set of simple methods that can help any scientist with treating the trajectory perhaps encouraging applying the involved methods. The involved methods will appear in software that we are developing now, helping therefore the experimentalists utilizing these methods on real data. Comparisons with other known methods in this field are made. [Formula: see text]Special Issue Comment: This article about mathematical treatments when solving single molecules is related to the reviews in this Special Issue about measuring enzymes67 and about FRET experiments2 and about the software QUB.6

scholarly journals Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions And Conformational Dynamics In Single-Molecule FRET

2018 ◽  
Author(s):  
Antonino Ingargiola ◽  
Shimon Weiss ◽  
Eitan Lerner
Keyword(s):  
Monte Carlo ◽  
Energy Transfer ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Distribution ◽  
Distribution Analysis ◽  
Distance Information ◽  
Additional Information

AbstractSingle-molecule Förster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Förster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances.Here we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.

Red light, green light: probing single molecules using alternating-laser excitation

2008 ◽  
Vol 36 (4) ◽  
pp. 738-744 ◽  
Author(s):  
Yusdi Santoso ◽  
Ling Chin Hwang ◽  
Ludovic Le Reste ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Dynamic Range ◽  
Red Light ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.

scholarly journals Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

2020 ◽  
Author(s):  
Martin F. Peter ◽  
Christian Gebhardt ◽  
Rebecca Mächtel ◽  
Janin Glaenzer ◽  
Gavin H. Thomas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Large Scale ◽  
Double Resonance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Resonance Spectroscopy ◽  
Distance Measurements ◽  
Single Molecule Fret

AbstractPulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Förster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

scholarly journals Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

2018 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Separation Distance ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Accuracy ◽  
Localization Microscopy ◽  
Signaling Receptors ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens1–5. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters6. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Förster resonance energy transfer (FRET) to quantify molecular interactions7. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

scholarly journals Single Molecule Characterization of Amyloid Oligomers

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 948
Author(s):  
Jie Yang ◽  
Sarah Perrett ◽  
Si Wu
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Amyloid Formation ◽  
Amyloid Aggregation ◽  
Wide Range ◽  
Amyloid Oligomers

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

scholarly journals FRETboard: semi-supervised classification of fret traces

2020 ◽  
Author(s):  
Carlos de Lannoy ◽  
Mike Filius ◽  
Sung Hyun Kim ◽  
Chirlmin Joo ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ground Truth ◽  
Optical Signals ◽  
Link Type ◽  
Wide Range ◽  
Classification Tool

AbstractFörster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nano-scale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner, but their operating system-dependent installation requirements and lack of flexibility impede wide-spread adoption. Here we propose to fit such models more efficiently and intuitively by adopting a semi-supervised approach, in which the user interactively guides the model to fit a given dataset, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios, and correctly estimate parameters for up to eleven states. On in vitro data we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.Availabilitysource code is available at https://github.com/cvdelannoy/FRETboard. The FRETboard classification tool is also available as a browser application at https://www.bioinformatics.nl/FRETboard.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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