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mutations), or intermolecular interactions. 1–4 In the past decade, more than 24 000 papers have been published using CD spectroscopy to characterise the structures of polypeptides and proteins. The information derived by CD spectroscopy, which can also include information on dynamic changes in solution or in environments such as membranes or films, is often complementary to that produced by other biophysical, computational and chemical methods such as crystallography, cryo-electron microscopy, NMR spectroscopy, FTIR spectroscopy, vibrational circular dichroism (VCD) spectroscopy, 5 molecular dynamics simulations, and low angle scattering. Indeed, in the same 10 year time period, nearly half of the publications which included CD spectroscopy also included information derived from at least one of these other techniques.ĬD spectroscopy has a number of advantages with respect to the higher resolution structural techniques such as crystallography, electron microscopy, and NMR spectroscopy in that it requires relatively small amounts of sample under conditions (temperature, concentration and components present) that may be more comparable to those found in cells. This has resulted in its wide-spread use both by the biochemical and structural biology communities to complement the information derived by those other biophysical methods, 4 as well as by the pharmaceutical industry, 6,7 to assess whether a protein is correctly folded, to monitor structural changes induced by interactions with ligands including other proteins, and to determine protein stability under environmental stresses induced by, for example, changes in pH or temperature. In recent years circular dichroism beamlines have been developed at synchrotron light sources, taking advantage of both their high light flux, which enables faster collection of data from smaller amounts of protein, and the higher information content available due to the lower wavelengths that can be achieved at these high-intensity light sources. This tutorial review discusses not only methods and software currently available for obtaining, processing, validating, and interpreting high quality CD data obtained using lab-based (commercial) CD instruments (as well as synchrotron radiation circular dichroism (SRCD) beamlines) on a variety of samples types, but also the bioinformatics tools and resources available for determining novel details of the structure and function of proteins based on such data. To optimise the amount of high quality and reproducible CD data obtained from a given sample, it is essential to follow good practice protocols for data collection (see Table 1 for example). 8,9 To accurately determine the secondary structure of a protein based on CD data, the data obtained must include a spectral range covering, at least, the wavelengths between 240 and 190 nm more accurate results are obtained if data collected includes even lower wavelengths, because more electronic transitions (peaks) will be included, increasing the information content of the data. To achieve such measurements, conditions must be used so that the total absorbance of the sample (due to protein, buffer and other added components) is below ∼1.2 at all wavelengths.