Accuracy of structural information obtained at the European Synchrotron Radiation facility from very rapid Laue data collection on macromolecules

The potential of very rapid Laue data collection for time-resolved studies down to the 150 ps timescale has been demonstrated in the case of cutinase, a 22 kDa lipolytic enzyme for which a considerable amount of structural information is available. This paper reports the derivation of the structure of native cutinase at 1.5 Angstrom from a Laue data set recorded at the White Beam Station of the European Synchrotron Radiation Facility (ESRF), with a total exposure time of 8.5 ns. The structure of the heteromorphous mutant R196E was chosen as a starting model for refinement, in order to check whether these fast Laue data were of sufficient quality to allow an accurate structure determination from a strongly biased starting model. This analysis is relevant because similar situations are encountered in fast time-resolved experiments where rapid structural modifications of a protein are analysed from fast Laue data sets, recorded in some excited states of the protein, and from a structural model representative of the rest state. 19 Laue images were recorded with 150 ps X-ray pulses emitted by a single electron bucket from the ESRF storage ring. With two insertion devices used in series, the available photon flux was sufficient to refine a satisfactory model of native cutinase (R-cryst = 19.3%; R-free = 24.2%). Discrepancies between this model and an accurate atomic model of cutinase (obtained from monochromatic data collected to 1.0 Angstrom resolution, R-cryst = 9.7%) were minor and mainly due to the nonoptimal completeness of the data (71.7% to 1.5 Angstrom) and to the different extent in resolution. The wild-type Arg196 could be readily positioned in the electron density and significant main- and side-chain displacements due to packing constraints were successfully retrieved with the Laue data. The electron-density maps were of sufficient quality to solve unambiguously these structural modifications. This feasibility study shows that very rapid Laue diffraction is a powerful tool to study protein dynamics in real time, provided that suitable macromolecular crystals as well as efficient reaction-triggering techniques are available.