AFM studies of the nucleation and growth mechanisms of macromolecular crystals

Atomic force microscopy (AFM) has been used to visualize events arising fro m the formation of intervening metastable phases at the surfaces of macromo lecular crystals growing from solution. Crystals investigated were of the p roteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of t ransfer RNA, and crystals of satellite tobacco mosaic virus. The appearance of aggregates on crystal surfaces was observed. The aggregates we infer to originate from liquid-protein droplets. These were particularly evident in freshly mixed mother liquor solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as crystall ine, multilayer stacks whose development was guided by, and contiguous with the underlying lattice. These contributed to the ordered growth of the cry stal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the unde rlying lattice, and these were subsequently incorporated into the growing s ubstrate crystal. Scarring experiments with the AFM tip indicated that, det ached from the crystal, molecules do not dissolve in the fluid phase but fo rm metastable liquid-protein droplets with a potential to rapidly crystalli ze on the crystal surface. The molecular structure of the growth steps for thaumatin and lipase protein crystals were deduced. There is no step roughn ess due to thermal fluctuations, and each protein molecule which incorporat ed into the step edge remained. Growth steps propagate by addition of indiv idual molecules which form subkinks of different size on the step edge. (C) 1999 Elsevier Science B.V. All rights reserved.