Protein crystallography in a vapour stream: data collection, reaction initiation and intermediate trapping in naked hydrated protein crystals

A procedure is presented for experiments on naked unfrozen protein crystals with the crystal mounted in a conventional cryo-loop and surrounded by a stream of a wet gas. The composition and temperature of the vapour stream can be adjusted to keep the crystal without deterioration for many hours. The arrangement allows (i) for rapidly testing crystals for diffraction before freezing, (ii) for data collection between 268–303 K with greatly reduced background, (iii) for the controlled drying or wetting of crystals, (iv) for the anaerobic manipulation of protein crystals, and (v) for the introduction of gaseous or volatile ingredients and reactants into the crystal. The technique offers new experimental possibilities,e.g.in time-resolved structural studies. Reaction initiation in many protein crystals can be achieved by changing the composition of the vapour stream to create a new chemical environment around the crystal and to introduce substrates/reactants either in the gas phase or as microdroplets. Spectral changes during such reactions can be monitored by single-crystal microspectrophotometry, and, once an intermediate has been detected at high concentrations, the crystal can be frozen,e.g.by rapidly switching the warm vapour stream to a cryogenically cooled helium or nitrogen jet. Representative examples are presented in this paper.