Journal of Crystal Growth 196 (1999) 489—502 AFM studies of the nucleation and growth mechanisms of macromolecular crystals Yu.G. Kuznetsov, A.J. Malkin, A. McPherson* Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA Abstract Atomic force microscopy (AFM) has been used to visualize events arising from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigated were of the proteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of transfer 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 crystalline, multilayer stacks whose development was guided by, and contiguous with the underlying lattice. These contributed to the ordered growth of the crystal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the underlying lattice, and these were subsequently incorporated into the growing substrate crystal. Scarring experiments with the AFM tip indicated that, detached from the crystal, molecules do not dissolve in the fluid phase but form metastable liquid-protein droplets with a potential to rapidly crystallize on the crystal surface. The molecular structure of the growth steps for thaumatin and lipase protein crystals were deduced. There is no step roughness due to thermal fluctuations, and each protein molecule which incorporated into the step edge remained. Growth steps propagate by addition of individual molecules which form subkinks of different size on the step edge.  1999 Elsevier Science B.V. All rights reserved. PACS: 81.10.Dn; 64.75.#g; 64.90.#b; 68.35.Bs Keywords: Protein crystals; Phase transitions; Atomic force microscopy; Nucleation; Crystal growth; Step structure 1. Introduction Colloidal systems, to an even greater degree than molecular systems, exhibit a particular richness in * Corresponding author. Fax: #1 949 8241954; e-mail: am- cphers@uci.edu. their number and variety of phases and phase transitions. Colloids, in addition to forming crystal- line solids, isotropic liquids (dense colloidal fluids) and gas phases (liquid phase), also assemble as plastic crystals, amorphous solids, glasses, liquid crystals, and gels. Considering the colloid particles as super-atoms interacting through a pair potential, which is not 0022-0248/99/$ — see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 9 8 ) 0 0 8 5 6 - 2