Kinetic cryocrystallography vs time-resolved crystallography - Nicolas Caramello скачать в хорошем качестве

Kinetic cryocrystallography vs time-resolved crystallography - Nicolas Caramello

Kinetic cryocrystallography vs time-resolved crystallography: when to trap reaction intermediates and when to observe them in real-time Two approaches exist for investigating systems out of equilibrium using crystallography. Historically, crystallographers have sought to halt or slow down reactions at specific steps by altering physicochemical parameters such as temperature or pH and using variants of their protein of interest. This first approach traps an intermediate of the reaction in the crystal long enough for crystallographic data to be collected under cryogenic conditions. Then, with brighter X-ray sources, it became possible to record diffraction patterns produced by brief X-ray pulses while a reaction was occurring in a crystal. This second approach generally relies on tuning the delay between reaction initiation and X-ray exposure to catch an intermediate state occurring in the crystal, close to physiological conditions, i.e at room temperature. Technical progress in the domain of serial crystallography at X-FELs and synchrotrons has made these time-resolved experiments more and more accessible, but valuable insight can still be gained from trapping experiments. In this presentation an overview of the different time-resolved crystallography methods available at X-FEL and synchrotron sources will be given, detailing what time domain and type of reaction they are suited for. The methods used to trap reaction intermediates in protein crystals will also be presented. Cases where trapping might be used synergistically with time-resolved crystallography will be discussed. Finally, the importance of characterising both trapped and caught crystal structures using complementary bio-physical methods also applicable to protein in solution, to check whether the reaction occurring in the crystals is physiological will be discussed.