In general, FRET allows measuring distances in the order of 30–80 Å, requires a low amount of material and is suitable to collect both structural (in steady-state measurements) and dynamic (in time-resolved OSI-744 concentration measurements) data. The disadvantage of the technique is that it requires bulky hydrophobic tags, limiting the positions where the fluorophores can be placed. At the same time the fluorescent tags might interact with the protein components of the complex, and either perturb the complex architecture or invalidate the assumption of low

fluorescence anisotropy. As an alternative approach to FRET, pulsed electron–electron double-resonance (PELDOR) spectroscopy can be used to determine distances in nucleic acids in the range of 15–70 Å. The method measures the dipole–dipole interaction of two free electrons located on nitroxide spin labels, chemically attached to the nucleic acid at selected positions [49]. Both distance and distance distribution functions can be obtained for double-labelled nucleotides [50]. The advantage of EPR-based distance measurement in comparison to FRET is that the spin labels are relatively

small (usually 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene, TPA) [42] and can be introduced both in helical and loop regions with minimal perturbation of the structure. In addition, the same spin labels can be employed for PRE measurements, optimizing the effort selleck chemical in engineering Cediranib (AZD2171) the spin label positions. Clearly a number of such long-range distances, obtained either by FRET or EPR, have the potential to restrict the conformational space available to the RNA and determine the relative orientation of both secondary structure elements in one RNA molecule and of multiple RNA molecules in the complex. In the past few years it has become popular to validate

or complement structural information obtained by NMR with Small Angle Scattering (SAS) data (Fig. 5). Small angle scattering of either X-ray (SAXS) or neutrons (SANS) provides a low-resolution envelope of the particle in solution. The structural information derived from SAS data refers to the overall shape of the molecule and does not report on fine structural details; in this respect it can be considered fully complementary to the information derived by NMR. Examples of the use of SAXS scattering profiles to validate structures derived by NMR can be found in the literature for both proteins [51] and nucleic acids [52] and [53]. Direct structural refinement against the SAXS scattering curve is available in the structure calculation program CNS [54]. Alternatively, SAXS data are used to derive a consensus low-resolution molecular shape: this shape can be employed to constrain the conformational space available to the molecule(s), similarly to the process of fitting flexible atomic structures to Electron Microscopy maps [55].