Collection and processing of X-ray diffraction data from protein crystals at high pressure

Apparatus for collecting X-ray diffraction data from protein crystals at pressures up to 0.2 GPa is described. The modified techniques required for crystal mounting, determination of crystal orientation in the beryllium pressure cell, and centering in the X-ray beam are described. Special attention is required for intensity absorption corrections due to the marked attenuation (90%) of the beam. Data have been collected from tetragonal hen egg white lysozyme crystals at 1000 atm to 2.0 Å nominal resolution using Cu Kα radiation (1 atm = 1.01325 × 10⁵ Pa). The crystals, crystallized in 0.86 M NaCl, require 1.4 M NaCl in the stabilizing solution to prevent cracking at several hundred atmospheres and higher. The crystals show no measurable hysteresis when cycled through several 1 to 1000 atm transitions. Altered intensities relaxed to their new value within 1 min on pressurization, but required about 90 rain to recover their starting values on pressure release. The unit-cell volume decreases 1.1% at 1000 atm.