New Trends in Macromolecular Crystallography at High Hydrostatic Pressure

Abstract

Publications on structures of macromolecules under high pressure determined by X-ray crystallography were limited until recently to two small monomeric proteins, hen egg-white lysozyme at 1 kbar (Kundrot and Richards, 1987) and sperm whale myoglobin at 1.5 kbar (Urayama et al., 2002). These studies were performed with a polycrystalline beryllium cell. Technical limitations were the low pressure limit (2 kbar), the lack of optical survey and noisy diffraction patterns due to scattering of X-rays by beryllium. Using the high pressure beamline ID30 at the ESRF, we achieved a technical breakthrough by the combination of a diamond anvil cell, ultra-short wavelength X-rays from undulators and a large imaging plate. The field of high-pressure macromolecular crystallography was extended both for the accessible pressure range (increased by nearly one order of magnitude) and data quality. Results obtained on tetragonal hen egg-white lysozyme crystals (tHEWL) at 7.0 kbar demonstrate that high quality diffraction data can be obtained under high pressure. In the case of tHEWL, data at 1.6 A resolution were measured on two crystals. The structure was redetermined at high pressure and refined. A detailed comparison between the 1.6 A structures at normal and high pressure is given. Another protein is under study, (Cu,Zn) superoxide dismutase (SOD). Good orthorhombic crystals were preserved beyond 12 kbar, which is noteworthy as the functional unit of SOD is a dimer. Data collection at 9 kbar is in progress. The following step is a preliminary study of cowpea mosaic virus crystal (CPMV), the first example of crystallized macromolecular assembly studied at high pressure. Oscillation pictures of a cubic crystal of CPMV were recorded at several pressures up to 4.4 kbar. The initial crystal (space group P23 at 1 bar, preserved at 1.1 and 2.0 kbar) was disordered and diffracted to low resolution. At 3.3 kbar, a highly ordered I23 crystal was obtained, which diffracts to 2.6 A resolution with high signal-to-noise ratio. At 4.4 kbar, the crystal no longer diffracted.