The authors thank B. Gruchmann for the mechanical drawings of the flow chamber. Further, we want to express our gratitude to Max Beckers and Florian Drechsler for insightful comments and discussions regarding NPS and the underlying sampling engine.

<ol>
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The authors have nothing to disclose.

We present the setup and experimental procedure to accurately determine FRET efficiencies between dyes attached via flexible linkers to biomacromolecules, i.e., nucleic acids and/or proteins.
In order to ensure precise smFRET measurements (Section 3), it is crucial to exclude air from the flow chamber at any time during the measurement. Furthermore, make sure to not overload the flow chamber with fluorophores. The fluorophores must be clearly separated to ensure correct analysis. As smFRET pairs, which do not show bleaching of the donor have to be excluded from the analysis, make sure that &#62;80% of the molecules in the field of view are bleached at the end of the movie. To account for inhomogeneities in the sample the &#946;-factor and the &#947;-factor, correcting the cross-talk and relative detection efficiencies of the donor and acceptor channel, respectively, are calculated for each FRET pair individually.
The camera settings (integration time, electron multiplier gain, pre-amplifier gain and readout rate described in Section 3.9) should be set to values giving the best tradeoff between signal to noise ratio, dynamic range and time-resolution. They need to be re-adjusted for different experiments or if different hardware is used. The numbers of frames need to be high enough to ensure that most of the donor molecules bleach within the observation time.
For the measurements on the fluorescence spectrometer (Sections 7 to 9) a good compromise between the signal intensity and the spectral resolution of the recorded data has to be found. To this end the slits in the excitation and emission pathway of the fluorescence spectrometer have to be adapted dependent on the instrument used and the sample concentration.
Moreover, we present the Fast-NPS analysis method to obtain structural information of transient or dynamic macromolecular complexes. NPS has been applied to reveal the path of the non-template DNA strand and the position of transcription initiation factors in the archaeal RNA polymerase open complex. Using the network of more than 60 different distance measurements, we showed that Fast-NPS, equipped with a newly implemented sampling engine (Eilert, T., Beckers, M., Drechsler, F., &#38; Michaelis, J. in preparation), reduces the time needed for the analysis of this complex smFRET network by &#8776;2 orders of magnitude, as compared to the original global NPS method27. The algorithm&#39;s robustness is rooted in a Metropolis-within-Gibbs sampler combined with a parallel tempering scheme. Fast-NPS shows exact reproducibility of network results and is consistent with results published earlier30.
Several different methods have been published aiming to infer structural information from smFRET measurements11,12,13,14,15,16,17,18. All of these approaches provide only one specific dye model. Thus, dyes, that do not fulfill the assumptions made by the respective model, cannot be used or lead to false structural information. Fast-NPS, on the contrary, allows to select for each dye molecule a different model. This helps to account for different conformational behavior of both, the dye molecule itself, as well as the linker used for its attachment. The local molecular surroundings of the dye molecule, as well as its physical properties will determine which model is most appropriate.
For the analyzed smFRET network of the archaeal initiation complex, an isotropic assumption for all dye molecules leads to a drastic decrease in the size of the credible volumes as compared to the classic model. In combination with a dynamic position averaging for all dye molecules the median of all credible volume sizes (at 95%) reduces to less than 0.5 nm3. However, these dye molecule posteriors are no longer consistent with their smFRET measurements, indicating that the assumptions made lead to false structural information. In contrast, the posteriors determined in the classic model are consistent with the determined smFRET efficiencies.
As the assumption of isotropic and/or dynamic position averaging for all dyes lead to inconsistencies, Fast-NPS enables dye molecule priors in which each dye can be assigned one of the five models. Each model uses the same accessible volume. The algorithm for the calculation of the dye AVs makes several assumptions. At first, the fluorophore&#39;s spatial shape is approximated by a sphere. Thus, a diameter taking into account the fluorophore&#39;s width, height and thickness should be used (Section 12). Further, the linker&#39;s shape is approximated by a flexible rod. The values presented in Section 12 were computed for the dye Alexa 647 attached via a 12-C linker. To date, it is not possible to accurately determine a priori which model is most suited, given an experimental geometry, and thus all models should be tested. In general, one will choose the model which gives the smallest possible posterior size, while still being consistent with the data. To test whether a choice of models is consistent with the smFRET data, we calculate both the posterior and the likelihood. Consistency means that more than 90% of the samples collected from the posterior are within the 95% confidence interval of the likelihood.
While it is true that the lower the anisotropy, the smaller the distance uncertainty, in a smFRET network geometric arrangements of the dye molecules also have to be taken into account. Thus, while representing dye molecules with a low fluorescence anisotropy with an iso model is a typical first choice, the consistency test provides a more direct means for selecting the correct dye model. The optimal choice of dye models can lead to a drastic increase in localization precision and at the same time retain the network&#39;s consistency with its FRET data.
To summarize, Fast-NPS allows to gain structural and dynamic information of large macromolecular complexes. In contrast to common structural methods such as x-ray crystallography or cryo electron microscopy this allows for monitoring highly flexible or transient complexes, thus greatly widening our mechanistic understanding of complex biological processes.

Determining the structure of a biomolecule is a key prerequisite for understanding its function. Two well-established methods for structure determination are cryo-electron microscopy and X-ray crystallography1,2. Today, both methods provide high-resolution structural information with a resolution down to the angstrom level. These two methods have been used extensively to elucidate the structure of large biomolecules such as protein complexes. Though the existing methods have constantly been improved throughout the last decades, the complexity of biological structures still poses a major challenge to structural biology, in particular when large, dynamic and transient complexes are investigated3.

In order to study the dynamics of macromolecular complexes and the structure-function relationship in particular, single-molecule methodologies have provided useful information4. Several new strategies were developed providing an orthogonal approach on acquiring structural and dynamic information. Examples are high speed AFM5, mechanical manipulation6, fluorescence localization microscopy7, as well as single-molecule F&#246;rster Resonance Energy Transfer (smFRET)8,9. Since quite early on FRET has been termed a molecular ruler, due to the distance dependence on the length scale of biomacromolecules10.
One particularly interesting application of smFRET is to use the distance information obtained from smFRET measurements to infer structural information11,12,13,14,15,16,17,18,19,20,21,22,23. Due to the high time resolution of smFRET, the position of mobile parts of a protein structure can be localized. However, in order to extract quantitative information from smFRET data important correction parameters about the dye molecules need to be determined during the measurement24. With these correction factors, the FRET efficiency EFRET can be calculated using the formula
<img alt="Equation 1" src="/files/ftp_upload/54782/54782eq1.jpg" />,
 
where IA and ID are the fluorescence intensities of the donor and the acceptor molecule, respectively (see Figure 2). The &#946;-factor accounts for cross-talk, the leakage of donor emission into the acceptor channel and is calculated by
<img alt="Equation 2" src="/files/ftp_upload/54782/54782eq2.jpg" />
where I&#39;A and I&#39;D are the fluorescence intensities of the donor and the acceptor molecule after photo bleaching of the acceptor molecule.
The &#947;-factor corrects the difference in the relative detection efficiencies in the two channels as well as the differences in the fluorescence quantum yield of the donor and the acceptor dye. It is calculated from every individual time trace by
<img alt="Equation 3" src="/files/ftp_upload/54782/54782eq3.jpg" />
Note, that this description neglects direct excitation of the acceptor molecule, which sometimes becomes important and would need to be corrected for as well. For determining these correction factors it is useful to excite both the donor as well as the acceptor in an alternating scheme25 in order to differentiate between photo-physical changes and structural dynamics.
In order to not only obtain quantitative smFRET efficiencies but also quantitative structural information, the Nano-Positioning System (NPS) was introduced in 200826. The name was chosen based on its similarities to the satellite-based Global Positioning System (GPS). The NPS is a hybrid technique combining smFRET and X-ray crystallography data for the localization of unknown dye positions in biomacromolecular complexes. The crystal structure serves as a reference frame and the smFRET results are used to obtain distance information between an unknown fluorophore position (antenna) and a position known from the crystal structure (satellite). In consecutive experiments the distances between the antenna and several satellites are measured and the position of the antenna is determined by means of a statistically rigorous analysis scheme based on Bayesian parameter estimation. As a result, not only the likeliest position of the antenna is computed, but its complete 3D uncertainty distribution, the so-called posterior, visualized by credible volumes. Moreover, NPS was expanded to allow for the analysis of complete smFRET networks27.
The NPS has been used to solve a number of important questions in eukaryotic transcription, namely the course of the upstream DNA, the non-template DNA and the nascent mRNA within the RNA Polymerase II elongation complex12,28, also demonstrating the effect of transcription initiation factors26 and the dynamic architecture of an open-promotor complex29. Moreover, the NPS was used to elucidate the structure of the archaeal RNA Polymerase open complex30 and in particular the position of transcription initiation factor TFE, which binds competitively to the same site as transcription elongation factor Spt4/531.
Since then, a number of smFRET based structural approaches have been published15,18,21,23. When comparing different smFRET based structural methods, it becomes clear that the apparent precision of the method is highly dependent on the particular choice of dye models. One should note that dye molecules may exhibit different spatial and orientational behavior depending on their local environment.
To this end, Fast-NPS was introduced32. Fast-NPS uses an advanced sampling algorithm reducing the calculation times drastically. Furthermore, Fast-NPS allows one to perform a structural analysis and for each dye molecule the user can choose from a set of five different dye models which will be described next. The most conservative model, called classic, assumes that the dye occupies only one, but unknown, position. At this position, the fluorophore can rotate freely within a cone, whose size is determined from its respective (time-dependent) fluorescence anisotropy. The orientation of the cone is not known, which leads to large uncertainties when converting measured smFRET efficiencies into distances. In this respect, the model is conservative, since it will lead to the smallest precision compared to the other dye models. Only for very short distances should the assumptions made by the classic model lead to a noticeably incorrect position determination. For typical smFRET values, the correct position is always enclosed in the comparatively large credible volume.
However, since a higher precision is desirable, it is important to develop and test alternative dye models, which could help to improve the precision. If the dye rotates much faster than its inherent fluorescence lifetime, the so-called iso model can be applied. Here, the orientation factor &#954;2 (needed for calculating the characteristic isotropic F&#246;rster radius<img alt="Equation 1" src="/files/ftp_upload/54782/54782eq4.jpg" />) is set to 2/3. As a result, the computed credible volumes are almost two orders of magnitude smaller as compared to those in the classic model32. In the case that the fluorophore is found in an environment that enables not only fast reorientation, but additionally fast motion all over its accessible volume, the meanpos-iso model should be used. In this model, the dye effectively occupies only one mean position, where the spatial averaging is accounted for by a polynomial distance conversion15. This model applies if for example the (commonly hydrophobic) dye is attached to a hydrophilic region, e.g., the DNA. Application of the meanpos-iso model leads to a further reduction in the size of the credible volumes by a factor of approximately two. However, a dye linked to a protein might bind reversibly to several hydrophobic patches in its sterically accessible volume (AV). A fluorophore that instantaneously switches between these regions, but within one region undergoes free rotation and fast localized motion is best described by the var-meanpos-iso model. For a similar situation in which the dye is not free to rotate the var-meanpos model applies. More details about these models can be found in our recent publication32.
These models provide an extensive repertoire to specifically account for the various environments a dye might encounter and applying them wisely optimizes its localization precision. In Fast-NPS every dye molecule attached to a specific position can be assigned to an individual model, such that FRET-partners are allowed to have different models. This enables limitless and close-to-nature modelling. However, it is important that one performs rigorous statistical tests to ensure that the result obtained by the final model combination is still in agreement with the experimental data. These tests are included in the Fast-NPS software.
In order to apply Fast-NPS to experimental data the measurement of (only) three input parameters is required. First, the dye-pair specific isotropic F&#246;rster radii (<img alt="Equation 1" src="/files/ftp_upload/54782/54782eq5.jpg" />) have to be determined. Therefore, the quantum yield (QY) of the donor dye, the donor fluorescence emission spectra and the acceptor absorption spectra need to be measured. These measurements can be carried out in bulk, using a standard spectrometer and a standard fluorescence spectrometer. For each pair, the R0 is then calculated using the freeware PhotochemCAD and can be used in the NPS analysis. Moreover, the (time-resolved) fluorescence anisotropies of the dye molecules need to be obtained using a polarization (and time) sensitive fluorescence spectrometer. However, the most important input parameters for Fast-NPS are the smFRET efficiencies measured on a single-molecule fluorescence microscopy setup, such as a total internal reflection fluorescence microscope (TIRFM).
Here, we present a step-by-step protocol for obtaining smFRET data and applying Fast-NPS (Figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Flowchamber preparation</td>
			<td style="width:167px;"></td>
			<td style="width:155px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Customized metall sample holder</td>
			<td style="width:167px;">self-built</td>
			<td style="width:155px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">quartz-glass slides, 76 x 26 mm</td>
			<td style="width:167px;">Technical Glass Products</td>
			<td align="right">26007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">coverslips, 60 x 24 mm</td>
			<td style="width:167px;">Marienfeld</td>
			<td align="right">101242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">detergent, Hellmanex II</td>
			<td style="width:167px;">Hellma</td>
			<td align="right" style="width:155px;">320.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ultra-pure water from Synergy UV</td>
			<td style="width:167px;">Millipore</td>
			<td align="right">2512600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zepto plasma cleaner</td>
			<td style="width:167px;">Diener</td>
			<td style="width:155px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">(3-aminopropyl)-triethoxysilane, p.a.</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:155px;">A3648</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">methoxy PEG-succinimidyl valerate, 5 kDa</td>
			<td style="width:167px;">Laysan Bio Inc.</td>
			<td>MPEG-SVA-5000-1g</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">biotinylated PEG-succinimidyl valerate, 5 kDa</td>
			<td style="width:167px;">Laysan Bio Inc.</td>
			<td>BIOTIN-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sodium biocarbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td>S5761 Sigma&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sodium carbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td>S2127 Sigma-Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">sealing film (Nescofilm)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td align="right">12981805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Tygon Flexible Silicone Tubing, 0.8 mm ID, 2.4 mm OD</td>
			<td style="width:167px;">Saint-Gobain Performance Plastics</td>
			<td align="right">720958</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Fine-Bore Polyethylene Tubing, 0.58 mm ID, 0.96 mm OD (Smiths Medical)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td align="right">12665497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Neutravidin</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:155px;">A2666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Total internal reflection fluorescence microscope</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Nd:YAG Laser, 532 nm</td>
			<td style="width:167px;">Newport Spectra-Physics</td>
			<td style="width:155px;">EXLSR-532-100-CDRH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">diode-pumped solid-state laser, 491 nm, Calypso</td>
			<td style="width:167px;">Cobolt</td>
			<td align="right" style="width:155px;">904010050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">diode laser 643 nm, iBeam smart</td>
			<td style="width:167px;">Toptica</td>
			<td style="width:155px;">iBEAM-SMART-640-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 532 RDC</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">F33-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 476 RDC</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">F33-476z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">acousto-optic tunable filter</td>
			<td style="width:167px;">AA Opto-Electronic</td>
			<td style="width:155px;">AOTFnC-VIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">plano-convex cylindrical lens, f = 75 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td>LJ1703L1-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">plano-concave cylindrical, f = -300 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">prism, PS 991</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PS991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">focussing lens, f = 75 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td>LA1608-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">syringe pump, PHD 2000</td>
			<td style="width:167px;">Harvard Apparatus</td>
			<td style="width:155px;">70-2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">2 stepper motors, Z812B</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">Z812B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">piezoelectric actuator, PE4</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PE4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">IR diode laser</td>
			<td style="width:167px;">Edmund Optics</td>
			<td style="width:155px;">CPS808</td>
			<td>part of the autofocus system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 775 DCXR</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">775 DCXR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">position-sensing detector (PSD), PDP90A</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PDP90A</td>
			<td>part of the autofocus system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">water-immersion objective, Plan Apo 60X WI, NA 1.2</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:155px;"></td>
			<td>MRD07601</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 645 DCXR</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">645 DCXR</td>
			<td>part of the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">emission filter, 3RD550-510</td>
			<td style="width:167px;">Omega Optical</td>
			<td style="width:155px;">3RD550-510</td>
			<td>green channel in the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">emission filter, 3RD660-760</td>
			<td style="width:167px;">Omega Optical</td>
			<td style="width:155px;">3RD660-760</td>
			<td>red channel in the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EMCCD camera, iXon+ DU897EBV</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;">AND-20-00032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EMCCD camera, iXon3 DU897D-BV</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;">AND-20-000141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Miscellaneous</td>
			<td style="width:167px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Varian 50</td>
			<td style="width:167px;">Cary</td>
			<td style="width:155px;"></td>
			<td>UV-VIS spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Fluorolog2</td>
			<td style="width:167px;">SPEX</td>
			<td style="width:155px;"></td>
			<td>fluorescence spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Solis (V4.15)</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;"></td>
			<td>control software for the EM-CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apt user utility&#160; (V1.022)</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td>control software for the piezo-motors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Norland Optical Adhesive 68</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td>adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC-AFN-0.8 Nile red</td>
			<td>Kisker Biotech</td>
			<td style="width:155px;"></td>
			<td>avidin-coated fluorescent multispec beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Matlab</td>
			<td style="width:167px;">Mathworks</td>
			<td style="width:155px;"></td>
			<td>technical computing language for custon written software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Origin (V9.0)</td>
			<td style="width:167px;">Originlab</td>
			<td style="width:155px;"></td>
			<td>scientific graphing and data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma 105-202-15-40</td>
			<td style="width:167px;">Hellma</td>
			<td>105-202-15-40</td>
			<td>absorption cuvette of 1 cm path length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma 105-251-15-40</td>
			<td style="width:167px;">Hellma</td>
			<td>105-251-15-40</td>
			<td>fluorescence cuvette with 3 mm path length</td>
		</tr>
	</tbody>
</table>

structural information from single-molecule fret experiments using the fast nano-positioning system

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

Transcription is the first step in gene expression in all organisms. In Archaea, transcription is carried out by a single RNA polymerase (RNAP). Compared to eukaryotes, the archaeal RNAP bears a striking structural resemblance to their eukaryotic counterparts while having a simpler transcriptional machinery. Thus, Archaea can be used as a model system to study eukaryotic transcription initiation by RNA Polymerase II (Pol II). Recently, the complete architecture of the archaeal RNA polymerase open complex has been determined from single-molecule FRET and NPS. The data from NPS analysis was used to build a model of the complete archaeal open promotor complex, which provides useful insights into the mechanism of transcription initiation.
To elucidate this structure, smFRET efficiencies were measured between unknown antenna dye molecules located within the open promotor complex and several known satellite dye molecules that were incorporated at five reference sites in the RNAP, whose positions are known from crystallographic structures (pdb-ID: 2WAQ)37. The antenna dyes were attached to either one of different positions on the non-template DNA, TFB, TBP or TFE. The complete network used in this study consisted of more than 60 measured distances.
Figure 7 depicts the model of the complete archaeal open promotor complex built from the NPS analysis. It comprises the double stranded promotor DNA (light and dark blue), the RNA Polymerase (grey) and the transcription initiation factors TBP (purple), TFB (green) and TFE (yellow). The model is superimposed with the results from the NPS analysis, the credible volumes, which were calculated using the classic model (A), the iso model (B), the meanpos-iso model (C), the var-meanpos-iso model (D) and the var-meanpos model (E).
<img alt="Figure 1" src="/files/ftp_upload/54782/54782fig1.jpg" /> 
Figure 1: Workflow of the acquisition and processing of the parameters needed for the Fast-NPS calculation. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54782/54782fig2.jpg" /> 
Figure 2: Exemplary fluorescence intensity time trace of a smFRET event. The fluorescence intensities of the donor (green) and the acceptor molecule (red) showing the three characteristic phases, namely I: smFRET, II: donor fluorescence after acceptor photobleaching, III: background fluorescence after donor photobleaching. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54782/54782fig3.jpg" /> 
Figure 3: Schematic illustration of the flow chamber for smFRET experiments. The flow chamber is mounted onto a customized metal holder with acrylic glass holders. The sandwich-design of the flow chamber comprises a quartz glass (fused silica) slide with two holes for attaching inlet and outlet tubing, a sealing film and a coverslip that closes the flow chamber. The prism for TIRF illumination is mounted onto the lower half of the flow chamber. Hollow tab screws provide the inlet and outlets for the flow chamber. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54782/54782fig4.jpg" /> 
Figure 4: Preparation of the quartz glass slide and the sealing film. Mechanical drawing of the quartz glass slide indicating the positions of the holes (given in millimeters). <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54782/54782fig5.jpg" /> 
Figure 5: Mechanical drawing of the flow chamber. The measures for the aluminum prism holder, acryl glass holders and aluminum mounting frame are given in millimeters. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54782/54782fig6.jpg" /> 
Figure 6: Schematic illustration of the prism-type TIRF setup used for smFRET experiments. Abbreviations for optical components: A, aperture; DM, dichroic mirror; F, emission filter; L, lens; M, mirror; O, objective; P, prism; PSD, position sensitive photo-diode; S, sample; PS, positioning stage; T, telescope. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54782/54782fig7.jpg" /> 
Figure 7: Simulation results of the different model assumptions. All pictures show the archaeal RNA polymerase (pdb-ID: 2WAQ, top view) together with the model for promoter DNA (tDNA and ntDNA in blue and cyan, respectively), TBP (purple), TFB (green) and TFE (yellow) in the archaeal open complex30. The credible volumes are superimposed for the NPS simulation results of (A) the classic model, (B) the iso model, (C) the meanpos-iso model, (D) the var-meanpos-iso model and (E) the var-meanpos model. All volumes are shown at 68% credibility. The classic and the var-meanpos networks are consistent with the smFRET data. In contrast, networks where for all dyes the iso, meanpos-iso or the var-meanpos-iso model is chosen are inconsistent with the measured data. <a href="http://cloudflare.jove.com/files/ftp_upload/54782/54782fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System at JoVE.com

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

structural-information-from-single-molecule-fret-experiments-using

Research

JoVE Journal

Biochemistry

12.1K Views.  Ulm University. We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Video: Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

gP2S, an Information Management System for CryoEM Experiments

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. 
The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

FRET Analysis of IDR Structural Sensitivity to Study Hyperosmotic Stress in Yeast

Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

Author Spotlight: Unlocking the World of Intrinsically Disordered Regions with Cellular Sensing and Responses

Intrinsically disordered regions (IDRs) are flexible protein domains that modify their conformation in response to environmental changes. Ensemble fluorescence resonance energy transfer (FRET) can estimate protein dimensions under different conditions. We present a FRET approach to assess IDR structural sensitivity in living Saccharomyces cerevisiae cells under hyperosmotic stress.

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical ...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.