W0321

Purification Improves Surface Morphology and Diffraction Resolution of Canavalin Crystals. Alexander J. Malkin^1,3, Marco Plomp¹, Bill R. Thomas², John S. Day¹, Alexander McPherson¹, and Alexander A. Chernov², ¹ Univ. of California, Irvine, Dept. of Molecular Biology and Biochemistry, 560 Steinhaus Hall, Irvine, CA 92697;² Universities Space Research Assn./NASA/MSFC SD46, Building 4481, MSFC, AL 35812; ³ BioSecurity and NanoSciences Laboratory, Dept. of Chemistry and Materials Science, Lawrence Livermore National Laboratory, Livermore CA 94551.

Homologous impurities are readily trapped by growing protein crystals and these impurities are known to induce strain stress, mosaicity, point and other defects. Thus, purification from these species was proposed to be a critical factor in limiting the resolution of X-ray diffraction analysis of macromolecular crystals and the reason why crystals of higher quality sometimes grow in microgravity. Here, canavalin was thoroughly purified by gel filtration HPLC prior to crystallization.

In situ AFM imaging shows that purification leads to a more regular crystal surface with polygonized steps (fig. b) compared to the surface with jagged steps for the non-purified solution (fig. a). Higher resolution AFM allowed to reveal species adsorbed on interstep terraces – potential step stoppers. Transformation of the jagged rounded to the regular polygonized spirals is interpreted as coming from low kink density on pure steps. X-ray diffraction data collected for several crystals show an average improvement in resolution by purification from 2.45 Å to 2.1 Å for I/σ = 1, proving that purification indeed is an important tool in improving X-ray diffraction resolution.