Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70 -75 degrees C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.

Atomic force microscopy was applied to investigate the influence of protein and precipitant (sodium-potassium tartrate) concentration on thaumatin crystal growth mechanisms. At constant protein concentration, a decrease of salt concentration from 0.8 to 0.085M caused a transition of the crystal growth mechanism from two-dimensional nucleation to dislocation growth. At different, fixed concentrations of salt, the protein concentration, which does not induce multiple crystal nucleation, was increased from 8 to 60mg/ml with corresponding increases in the tangential velocity of growth steps from 5 to 17.5nm/s. Results from these experiments suggest that a highly concentrated protein solution, as might be found in a protein rich phase, may not induce crystal nucleation, but can promote crystal growth if screw dislocations are present in the crystal. © 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), cucumber mosaic virus (CMV) and glucose isomerase. Growth of these crystals proceeded by two-dimensional (2D) nucleation. For glucose isomerase, from supersaturation dependencies of tangential step rates and critical step length, the kinetic coefficients of the steps and the surface free energy of the step edge were calculated for different crystallographic directions. The molecular structure of the step edges, the adsorption of individual virus particles and their aggregates, and the initial stages of formation of 2D nuclei on the surfaces of TYMV and CMV crystals were recorded. The surfaces of individual TYMV virions within crystals were visualized, and hexameric and pentameric capsomers of the T = 3 capsids were 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 cour se of recording the in situ development of the TYMV 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. © 2001 Elsevier Science B.V. All rights reserved.

For the first time, virus crystal growth dynamics and morphology have been investigated in real time on the nanometer scale. Individual monomers on the (111) face of cubic satellite tobacco mosaic virus (STMV) crystals were resolved and used to determine crystal packing. Growth of STMV proceeded by two- and three-dimensional nucleation to formed "stacks" of islands. No dislocations were observed. Small islands provided an estimate of critical radius size and the free energy of the step edge, α. Step advancement rates were used to determinate the kinetic coefficient β. Images illustrate mechanisms for defect incorporation and suggest factors that limit growth rate and uniformity. © 1995 The American Physical Society.

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 crystal's diffraction pattern.

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.

Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.

A chimeric human-simian IgG, antigen specific for CD4, when exposed to 0.5 M SO(=)(4) containing 0.4% polyethylene glycol or Jeffamine, self-assembles into discreet, roughly spherical particles 23 nm in diameter. Increasing SO(=)(4) to 1.55 M induces the IgG particles to crystallize in either a hexagonal or a monoclinic form. From X-ray diffraction, the former crystal is of space group P622, with one IgG particle in the unit cell; thus the particle itself must have 622 point group symmetry. Both crystal forms have been imaged using atomic force microscopy. Detailed features of the duodecamer were evident, including the symmetry and a large solvent channel along the sixfold axis. The particles in some ways resemble the hexameric IgG aggregates believed to activate compliment upon antigen binding and, therefore, may have physiological relevance. Investigation of seven other IgGs of diverse origins and subclasses indicates that many, if not most, IgGs form similar particles. To our knowledge, this is the first observation of the assembly of IgG into high symmetry aggregates in the absence of antigen or their crystallization.

Transient photoconductivity (σph) experiments have been carried out in single crystals of insulating La2CuO4+δ near the metal-insulator transition (δ≈ 1-2%). The time evolution of the σph changes dramatically with light intensity (IL). At low IL, σph is characterized by power law time decay, t-α, in the nanosecond time regime, and the exponent, α, decreases significantly with increasing IL. As IL increases to 5 × 1015 photons/cm2, σph reaches ∼ 15 S/cm, and the lifetime of the conducting state is enhanced by more than two orders of magnitude: σph exhibits a delayed peak centered at approximately 30 ns. followed by exponential decay with time constant of ∼ 360 ns. The data reveal a continuous distribution of localized electronic states above the (hole) mobility edge; the M-I transition is achieved when the Fermi level is shifted away from the localized states and across the mobility edge either by chemical dopiing or by photo-excitation. The time of σph at high IL implies the formation of metastable metallic droplets after photo-excitation. © 1992.

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.