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In situ atomic force microscopy has been used to investigate step dynamics and surface evolution during the growth of single crystals of canavalin, a protein with a well known structure. Growth occurs by step flow on complex dislocation hillocks, and involves the formation and incorporation of small, mobile molecular clusters. Defects in the form of hollow channels are observed and persist over growth times of several days. The results are used to establish a physical picture of the growth mechanism, and estimate the values of the free energy of the step edge, n, and the kinetic coefficient, P.PACS numbers: 87.15. Da, 61.16.Ch, 61.50.Cj, 68.35.Bs The crucial functions of biological systems are governed by macromolecules, such as proteins and nucleic acids, whose specific roles are in turn defined by their structures.Experimentally, macromolecular structure is determined using x-ray crystallographic methods [1,2] which require large, uniform single crystals of the macromolecules. However, crystallization of proteins and other biological molecules is highly problematic, to such an extent that it has become the rate limiting step in most structure analyses. This is true, in large part, because little is know of the growth mechanisms, the events leading to nucleation, or the fundamental thermodynamic and kinetic parameters that determine growth rates and surface morphologies. Less still is understood about the incorporation of defects, the forces responsible for the orientation and bonding of molecules in the lattice, or the role of transport processes, a factor of particular relevance to studies of the phenomenon in microgravity environments [3,4]. A more detailed and comprehensive understanding of all these questions is required if the current obstacles to macromolecular crystal growth are to be overcome.Beyond this important practical consideration is the realization that the slow growth kinetics and large molecular diameters of biological molecules make macromolecular crystals ideal systems for the investigation of homoepitaxy in real time using scanned probe microscopics. While real time experiments have been performed using scanning tunneling microscopy on both metal [5] and semiconductor [6,7] Fig. 1(a) and 1(b). The canavalin trimer forms a three-sided ring with an outside diameter of 8.6 -8.8 nm, an inside diameter 1.8 nm, and a thickness of 3.5 -4.0 nm. As Fig. 1(b)

For the X-ray crystallographer, the future of his field is, and has always been, determined by what he is able to crystallize. This is now particularly true as a result of the extraordinary improvement in X-ray detectors and sources [1], the advent of computers of amazing speed, and the development of programs that are both friendly and efficient [2]. The critical component, trailing other technology, is the growth of crystals of macromolecules having sufficient size and quality to permit X-ray analysis.Crystallization, however, has become less problematic. With synchrotron sources [3], sensitive, fast detectors, cryogenic techniques that eliminate radiation damage [4], and more powerful phasing tools, both the number and the size of crystals required for the analysis has decreased: crystals of 30-50 ,pm may soon be adequate, frozen crystals often provide a complete data set, and molecular replacement can allow determination of entire structures from this one set. Equally importantly, crystallization procedures, reagents, and diagnostic tools are now available that greatly improve the probability of success for both experienced crystallographers and interested biochemists.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).