High-pressure protein crystallography (HPPX):: instrumentation, methodology and results on lysozyme crystals

A new set-up and associated methodology for the collection of angle-dispersive diffraction data from protein crystals submitted to high hydrostastic pressure have been developed on beamline ID30 at the ESRF. The instrument makes use of intense X-rays of ultra-short wavelength emitted by two collinear undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffraction pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) and orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase (SOD) were recorded at room temperature and pressures up to 0.915 and 1.00 GPa, respectively. The compressibility of tHEWL was determined from unit-cell parameters determined at 24 different pressures up to 0.915 GPa. High-pressure diffraction data sets from several crystals of tHEWL were collected and analyzed. Merging of data recorded on different crystals at 0.30 and 0.58 GPa produced two sets of structure amplitudes with good resolution, completeness, redundancy and R-sym values. A third set at 0.69 GPa was of a similar quality except a lower completeness. The three structures have been refined. The pressure-induced loss of crystalline order in a tHEWL crystal beyond 0.82 GPa was captured through a series of diffraction pictures.