Abstract
The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for `spectroscopy'). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.
Highlights
Performing optical spectroscopy on crystals, or in crystallo optical spectroscopy, has progressively matured as a technique complementary to protein crystallography
Many enzymes rely on cofactors that absorb in the nearUV/blue part of the light spectrum
While it was rapidly realised that the chromophores of fluorescent proteins, as well as the surrounding residues, were sensitive to radiation damage by X-rays (Adam et al, 2009; Royant & Noirclerc-Savoye, 2011; Figs. 6h and 7h) and that special care is required during data collection and structural analysis, the Cryobench has become an essential instrument to study the structure and function of proteins homologous to green fluorescent protein (GFP; Tsien, 1998)
Summary
Performing optical spectroscopy on crystals, or in crystallo optical spectroscopy (icOS), has progressively matured as a technique complementary to protein crystallography. While the most common application of icOS is the assessment of whether a crystallized protein is in a similar functional state as in solution (Bourgeois & Royant, 2005; Pearson et al, 2004), the technique has become of great help in monitoring the level of X-ray-induced damage (or ‘radiation damage’) occurring during crystallographic experiments on bright synchrotron beamlines (Garman, 2010). Crystallographic methods to determine the structure of unstable protein species, either as a function of time (reaction-intermediate state) or of X-ray dose (X-ray-sensitive state), have been named kinetic crystallography (KX; Bourgeois & Royant, 2005). We describe the third version of the ESRF Cryobench installation, summarize improvements compared with the previous version, review the various types of proteins studied and the types of experiments carried out using icOS at the ESRF and comment on experimental difficulties
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