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Accuracy of Structural Information Obtained at the European Synchrotron Radiation Facility from Very Rapid Laue Data Collection on Macromolecules
Author(s) -
Bourgeois D.,
Longhi S.,
Wulff M.,
Cambillau C.
Publication year - 1997
Publication title -
journal of applied crystallography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.429
H-Index - 162
ISSN - 1600-5767
DOI - 10.1107/s0021889896010862
Subject(s) - cutinase , synchrotron radiation , monochromatic color , chemistry , crystallography , physics , optics , enzyme , biochemistry
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 ( 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 Å, resolution, R cryst = 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.