title: Colouring cryo-cooled crystals: online microspectrophotometry.
abstract: X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200-1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.
authors.affiliation: EMBL, 6 rue Jules Horowitz, 38042 Grenoble, France.
authors.forename: John
authors.initials: J
authors.lastname: McGeehan
authors.forename: Raimond B G
authors.initials: RB
authors.lastname: Ravelli
authors.forename: James W
authors.initials: JW
authors.lastname: Murray
authors.forename: Robin Leslie
authors.initials: RL
authors.lastname: Owen
authors.forename: Florent
authors.initials: F
authors.lastname: Cipriani
authors.forename: Sean
authors.initials: S
authors.lastname: McSweeney
authors.forename: Martin
authors.initials: M
authors.lastname: Weik
authors.forename: Elspeth F
authors.initials: EF
authors.lastname: Garman
mesh_terms: D003080:Cold Temperature; D003116:Color; D017076:Computer-Aided Design; D003460:Crystallization / Q000379:methods*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D009862:Online Systems; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation* / Q000379:methods; D014961:X-Ray Diffraction / Q000295:instrumentation* / Q000379:methods