ISSN:
1600-5767
Source:
Crystallography Journals Online : IUCR Backfile Archive 1948-2001
Topics:
Geosciences
,
Physics
Notes:
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 Å 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, tile available photon flux was sufficient to refine a satisfactory model of native cutinase (Rcryst = 19.3%; Rfree = 24.2%). Discrepancies between this model and an accurate atomic model of cutinase (obtained from monochromatic data collected to 1.0 Å, resolution, Rcryst = 9.7%) were minor and mainly due to the nonoptimal completeness of the data (71.7% to 1.5 Å) 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1107/S0021889896010862
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