Yu. G. Kuznetsov

Imaging of viruses by atomic force microscopy

Atomic force microscopy (AFM) has been used to image a variety of virus particles in vitro and could, conceivably, be used as a useful diagnostic for their presence, their structural characterization and even their identification. Virus particles can be imaged by AFM in air, under alcohol or in physiological medium, and accurate measurements of their dimensions obtained. In addition, the appearance and organization of capsomere structures on their surfaces are frequently visible. A number of viruses and virus crystals have been imaged successfully using AFM and improvements in AFM technology and sample handling will undoubtedly increase even more its power, resolution and scope.

AFM studies of the nucleation and growth mechanisms of macromolecular crystals

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.

Incorporation of impurities into macromolecular crystals

The chemical, mechanical and diffraction properties of crystals grown from solution, as well as their growth kinetics and morphological development, depend very much on the types and concentrations of impurities present in their mother liquor. The situation appears vastly more complicated in the case of macromolecular crystals because of the complex nature of the molecules and the biochemical milieu from which they are derived. An attempt is made here to catalog and characterize these various impurities. One class of impurities, large foreign particles (such as dust), microcrystals, misoriented three-dimensional nuclei, and large molecular clusters has been investigated in detail using atomic force microscopy. With this technique we have directly visualized the incorporation of such larger impurities and have delineated some of their more striking consequences. In particular we have found that in some cases such incorporation is accompanied by visible defect formation or dislocations. In other cases of small three-dimensional nuclei, coalescence proceeds in a smooth manner, with alignment and knitting together of the respective lattices. A calculation of the overall defect density in canavalin crystals shows the number of defects to be many orders of magnitude greater than found for most conventional crystals.

Macromolecular crystal growth as revealed by atomic force microscopy

Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. We can, using AFM, image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development. All of these can be recorded under normal growth conditions, in native mother liquors, over time intervals ranging from minutes to days, and at the molecular level.

Atomic force microscopy applications in macromolecular crystallography

Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non-perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystals diffraction pattern.

In situ atomic force microscopy studies of protein and virus crystal growth mechanisms

In situ atomic force microscopy was utilized in studies of the growth mechanisms of six protein and virus crystals. It was demonstrated that those macromolecules utilize all of the growth mechanisms found in the crystallization of conventional molecules. These include growth on screw dislocations and by two- and three-dimensional nucleation. In addition the mechanism of normal growth was observed as well. Estimates for the fundamental parameters (free energy of the step edge, α, and kinetic coefficient of steps, β) for the crystallization of some macromolecules were obtained and compared with those known for inorganic crystals grown from solution.

An in situ AFM investigation of catalase crystallization

Surface morphology, growth and dissolution of crystals of the protein catalase were studied by in situ atomic force microscopy (AFM). Growth of the (001) face of catalase crystals proceeds in alternating patterns by two-dimensional nucleation and successive deposition of two distinctive growth layers, each having a step height equal to half the unit cell parameter. Shapes of two-dimensional nuclei exhibit strong asymmetry due to directional anisotropy in step rates. The shapes of islands on successive layers are related by two-fold rotation axes along the 〈001〉 direction. Lattice resolution AFM images of the molecular structure of sequential surface layers were recorded. Adsorption of large three-dimensional clusters of molecules was also observed to occur on the surfaces of catalase crystals. These clusters developed into either properly aligned multilayer stacks or misaligned microcrystals. Incorporation of misoriented microcrystals as large as 50 × 3 × 0.1 μ m3 proceeded without formation of defects. Upon incorporation of microcrystals and subsequent deposition of new layers on the surface of a growing crystal, impressions with depths of up to 0.4 of the growth layer thickness formed due to misfits between the lattices of the microcrystals and those of the growth layers. This produced elastic deformations in growth layers of ≈0.6%.

Atomic force microscopy studies of icosahedral virus crystal growth.

Biological macromolecules and particularly viruses, provide excellent systems for the study of crystallization from solution because of their relatively large size. The kinetics of their crystallization is at least an order of magnitude less than for conventional systems, and their large size permits visualization, both of crystal lattices and individual particles, by techniques such as atomic force microscopy (AFM). This technique is especially powerful for biological macromolecules because it can be utilized in situ, in the crystallization mother liquor, over long periods of time without perturbing the growing crystals. We present here observations using AFM of the nucleation and growth of crystals of satellite tobacco mosaic virus, and some recordings as well of bromegrass mosaic virus. Correlations are made, where possible, with corresponding analyses using X-ray diffraction analysis.

Michelson interferometric studies of protein and virus crystal growth

Abstract In situ laser Michelson interferometry was utilized to investigate the growth kinetics and surface morphology in canavalin, thaumatin, and turnip yellow mosaic virus (TYMV) crystallization. Interferometric patterns and kinetic measurements from growing macromolecular crystals as small as 20 μm were obtained. This study shows that for the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces of growing crystals. Supersaturation dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were determined. The kinetic coefficient β was estimated for canavalin grown from two different precipitant systems to be 3.2 × 10 −4 and 5.3 × 10 −4 cm s −1 , respectively. The change in activities of dislocation sources under different growth conditions was analyzed.

The incorporation of large impurities into virus crystals

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.