Minimal Resolution

The determination of macromolecular structures by X-ray crystallography at a level of detail sufficient for the construction of reliable atomic models requires crystals that diffract X-rays by 3.5 A2 or better. A resolution of at least 3.5 A is required to see structural elements in proteins,

2 1 Ä (Angstrom) = 0.1 nm = 10"'° m such as alpha-helices or beta-sheets, which can be directly visualized in electron density maps calculated using the X-ray data.

Where the general fold of the protein chain is desired, an analysis at 3.5 Â may suffice to determine the protein structure. However, at this resolution the orientation of hydrogen bonding groups is not well determined, and detailed questions regarding the structural architecture of the protein cannot be answered reliably until a resolution of ~2.5 Â or better is achieved. The precise calculation of the energetics of ligand binding or intermolecular interfaces requires structure determination carried out to an even higher resolution, making possible the mapping of ordered water molecules and an accurate description of hydrogen bonding geometries (Geierstanger et al. 1996). The most accurate protein structure determinations are carried out at a resolution of 1.5 Â or better. In the relatively rare cases where data to better than 1 Â are obtained, individual hydrogen atoms can often be distinguished and the disorder within the protein structure can be described in detail.

The intrinsic resolution of a protein crystal can be thought of as arising from two factors. One is the mosaicity, a parameter that is a measure of the misalignment between small coherent blocks of individual molecules within the larger crystal. While crystals that are highly mosaic may diffract to high resolution, the high mosaicity leads to a broadening of the diffraction spots, which can complicate or even foil their measurement. The other crystal characteristic that affects resolution is the Debye-Waller Factor, also known as the overall temperature factor, which reflects disorder and mobility within the individual molecules that make up the crystal.

Many protein molecules that are of interest today, particularly those that are studied in the form of relatively unstable complexes, are expected to have intrinsically high Debye-Waller factors, limiting the resolution of the resulting diffraction pattern. In such cases, if the size of the perfectly aligned mosaic blocks can be increased, the resulting increase in the sharpness of the diffraction pattern can effectively improve the resolution of the diffraction pattern. In such situations, if growth in microgravity produces crystals with larger mosaic blocks (lower mosaicity), then there may be a significant improvement in the quality of the diffraction measurements. These added levels of detail would enable researchers to see the functional groups and water molecules and thereby more fully understand the interactive mechanisms of macromolecular assemblies (Chayen and Helliwell 1999).

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