A nano-positioning system for macromolecular structural analysis

Abstract

Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5¢ end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.In recent years, high-resolution structural models of large macromolecular complexes such as the ribosome 1 , the RecBCD helicase 2 or RNA polymerases 3,4 have been obtained using X-ray crystallography. Although these structures provide detailed insight into the molecular architecture of complex biological systems, the position of flexible domains can usually not be determined because of averaging effects.Single-molecule methods, on the other hand, provide the possibility of directly obtaining structural information because they allow the study of real-time conformational changes of macromolecular complexes 5 . In combination with fluorescence resonance energy transfer (FRET) 6 , a technique that has been termed a molecular ruler 7 , one can in principle measure distances within a macromolecule in real-time. However, because of experimental problems such as variations in quantum yield 8 or dependence of FRET on the orientations of the two dye molecules 9 , there are few examples in the literature of quantitative distance measurements 8,10-12 or position determination 13-16 using single-pair FRET (sp-FRET). Instead, these data are more often interpreted in a qualitative fashion monitoring conformational changes and length increases or decreases [17][18][19][20] .Using triangulation of several FRET distance measurements, it is possible to determine a previously unknown position [13][14][15][16][21][22][23] . Although these experiments are able to estimate the most likely position, they do not show how existing experimental uncertainties might affect the position determined. Therefore, these positions must be interpreted with great caution because one has no information about the experimental accuracy. In principle, one can conduct control measurements that provide validity tests of the position determined 14 , but to arrive at a quantitative technique, experimental uncertainties must be taken into account.Here we used bayesian parameter estimation 21 , a probabilitybased analysis method, to compute the three-dimen...

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

“…Therefore, the variance of the FRET efficiency distribution in the TD-A1 experiment is much smaller for the tetramer than for the dimer. Several groups have recently developed tools to calculate an accurate distribution of the distance between the donor and acceptor by appropriate modeling of fluorophores and linkers for macromolecules with well-defined structures (64)(65)(66)(67)(68)(69). This calculation may allow for a more quantitative comparison.…”

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

“…Several methods have been developed to account for this uncertainty. These methods include Bayesian statistical methods and molecular dynamics modeling to calculate a region that the fluorophore attached to the protein will likely occupy during the excited-state lifetime (30,31). Although these computational methods improve the ability to estimate the location of the fluorophore in space, an experimental method that precludes the use of computation to find backbone positions would be advantageous.…”

Single molecule studies of RNA polymerase II transcription in vitro

“…The data are analyzed using Bayesian parameter estimation to generate a probability density function for the unknown position (i.e., the position of the antennae dye molecule). 16 Yeast RNAP II open complexes containing TBP, TFIIB, TFIIF, RNAP II, and heteroduplex DNA (i.e., contains an engineered bubble around the transcription start site) were studied using a NPS. 15 Downstream DNA in open complexes was found to fluctuate between two locations: within and above the RNAP II cleft.…”

“…18 This discrepancy could potentially arise from differing experimental conditions, such as ion concentrations. 19 Interestingly, a NPS study found that in the absence of TFIIB, exiting RNA was more likely to contact the dock domain of RNAP II, but upon addition of TFIIB, the contact shifted toward the Rpb4-Rpb7 subunits, 16 suggesting that this switch could be regulated during early transcription.…”