In situ atomic force microscopy was utilized to study the relation between crystal structure, surface morphology, and growth mechanisms of faces of macromolecular crystals that have screw axes perpendicular to them. It was found that the {001} faces of orthorhombic catalase, trigonal trypsin, and tetragonal Bence-Jones protein (BJP) crystals grow by successive deposition of n alternating, symmetry-related layers with a thickness of d0 0(n)=1/n|c|. These layers are associated with two-, three- and four-fold screw-axes in the crystal structure of catalase (n=2), trypsin (n=3) and BJP (n=4) crystals, respectively. Implications for growth rates and three-dimensional morphological development are discussed. © 2002 Elsevier Science B.V. All rights reserved.

A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in "salting out" or "salting in' procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The "liquid protein" phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase. © 2001 Elsevier Science B.V. All rights reserved.

In situ atomic force microscopy (AFM) was used to investigate surface evolution during the growth of single crystals of turnip yellow mosaic virus (TYMV). Growth of the (101) face of TYMV crystals proceeded by two-dimensional nucleation. The molecular structure of the step edges and adsorption of individual virus particles and their aggregates on the crystalline surface were recorded. The surfaces of individual virions within crystals were visualized and seen to be quite distinctive with the hexameric and pentameric capsomers of the T = 3 capsids being clearly resolved. This, so far as we are aware, is the first direct visualization of the capsomere structure of a virus by AFM. In the course of recording the in situ development of the crystals, a profound restructuring of the surface arrangement was observed. This transformation was highly cooperative in nature, but the transitions were unambiguous and readily explicable in terms of an organized loss of classes of virus particles from specific lattice positions. In some cases areas of a single crystal surface were recorded in which were captured successive phases of the transition. We believe this provides the first visual record of a cooperative restructuring of the surface of a supramolecular crystal.

Virus crystals can incorporate a wide range of unusual impurities, not possible for conventional crystals, or even most protein crystals because of the large size of their constituent particles. These impurities include anomalous virions, satellite viruses and biological fibers. Examples of several of these unusual impurities are presented here, along with some of the consequences for the crystal lattices. The high solvent content, the forgiving character of the lattice and the plasticity of the virions allow these incorporations to be possible.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.

A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles. Atomic force microscopy can thus be used as a diagnostic tool to provide a rapid way to obtain high-resolution images of human pathogens from crude preparations. It is a useful technique that complements X-ray-based structure determination, cryo-electron microscopy techniques, and optical microscopies in the field of molecular pathogenesis.