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

A new set-up and associated methodology for the collection of angle-dispers ive diffraction data from protein crystals submitted to high hydrostastic p ressure have been developed on beamline ID30 at the ESRF. The instrument ma kes use of intense X-rays of ultra-short wavelength emitted by two collinea r undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffrac tion pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) an d orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase ( SOD) were recorded at room temperature and pressures up to 0.915 and 1.00 G Pa, respectively. The compressibility of tHEWL was determined from unit-cel l parameters determined at 24 different pressures up to 0.915 GPa. High-pre ssure diffraction data sets from several crystals of tHEWL were collected a nd analyzed. Merging of data recorded on different crystals at 0.30 and 0.5 8 GPa produced two sets of structure amplitudes with good resolution, compl eteness, redundancy and R-sym values. A third set at 0.69 GPa was of a simi lar quality except a lower completeness. The three structures have been ref ined. The pressure-induced loss of crystalline order in a tHEWL crystal bey ond 0.82 GPa was captured through a series of diffraction pictures.