Macromolecular crystal growth investigations using 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. It is possible, using AFM, to 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.

Structure of intracellular mature vaccinia virus visualized by in situ atomic force microscopy.

Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended approximately 16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.

Repair of impurity-poisoned protein crystal surfaces.

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.

Application of atomic force microscopy to studies of surface processes in virus crystallization and structural biology.

Atomic force microscopy (AFM) investigation revealed the sources of disorder and mechanisms of their formation in crystals of an icosahedral plant virus, Cucumber Mosaic Virus (CMV) and structure of the Herpes Simplex Virus (HSV-1). The combination of defects and local disorder in CMV crystals presented here are likely the physical bases for mosaicity in virus crystals, and may be largely responsible for their limited diffraction resolution. High-resolution images of intact, enveloped HSV-1 and the underlying capsid structure demonstrate capabilities of AFM to probe structures of large macromolecular assemblies.

Self-repair of biological fibers catalyzed by the surface of a virus crystal.

Helical fibers, presumably proteinaceous and of microbial origin, have been visualized by atomic force microscopy on the surfaces of crystals of satellite tobacco mosaic virus. If the crystals are growing, then the fibers are incorporated intact into the crystal lattice. If broken on the crystal surface, then within a few minutes, the fibers self-reassemble to reestablish continuity. This, we believe, is the first observation of such a crystal surface-catalyzed repair of a biological structure. The surfaces of virus crystals provide ideal workbenches for the visualization and manipulation of nanoscale objects, particularly extended structures such as these fibers.

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 crystal's diffraction pattern.

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin.

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

Atomic force microscopy in the study of macromolecular crystal growth.

Atomic force microscopy (AFM) has been used to study protein, nucleic acid, and virus crystals in situ, in their mother liquors, as they grow. From sequential AFM images taken at brief intervals over many hours, or even days, the mechanisms and kinetics of the growth process can be defined. The appearance of both two- and three-dimensional nuclei on crystal surfaces have been visualized, defect structures of crystals were clearly evident, and defect densities of crystals were also determined. The incorporation of a wide range of impurities, ranging in size from molecules to microns or larger microcrystals, and even foreign particles were visually recorded. From these observations and measurements, a more complex understanding of the detailed character of macromolecular crystals is emerging, one that reveals levels of complexity previously unsuspected. The unique features of these crystals, apparently in AFM images, undoubtedly influence the diffraction properties of the crystals and the quality of the molecular images obtained by X-ray crystallography.

Surface processes in the crystallization of turnip yellow mosaic virus visualized by atomic force microscopy.

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.

Structure of orthorhombic crystals of beef liver catalase.

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the <001> direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the 'bottoms' and 'tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Visualization of RNA crystal growth by atomic force microscopy.

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.

Molecular resolution imaging of macromolecular crystals by atomic force microscopy.

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).

Atomic force microscopy studies of living cells: visualization of motility, division, aggregation, transformation, and apoptosis.

Atomic force microscopy, in contact mode, has been used to image living mammalian cells in culture at both low and high resolutions. The method is shown practical for revealing cytoskeletal features beneath the cell membrane and their restructuring during a variety of cellular activities. Among the processes that we have visualized are locomotion, tissue formation, cell division, transformation by viruses, and cell death. We show that some processes that occur well within cells can, nonetheless, be observed using the atomic force probe. At high resolution, features on the cell surface on the order of 0.5 micron and their changes with time, can be recorded.

Incorporation of microcrystals by growing protein and virus crystals.

In the course of time-lapse video and atomic force microscopy (AFM) investigations of macromolecular crystal growth, we frequently observed the sedimentation of microcrystals and three-dimensional nuclei onto the surfaces of much larger, growing protein or virus crystals. This was followed by the direct incorporation over time of the smaller crystals into the bulk of the larger crystals. In some cases, clear indications were present that upon absorption of the small crystal onto the surface of the larger, there was proper alignment of the respective lattices, and consolidation proceeded without observable defect formation, i.e., the two lattices knitted together without discontinuity. In the case of at least one virus crystal, cubic satellite tobacco mosaic virus (STMV), addition of three-dimensional nuclei and subsequent expansion provided the principal growth mechanism at high supersaturation. This process has not been reported for growth from solution of conventional crystals. In numerous other instances, the lattices of the small and larger crystals were obviously misaligned, and incorporation occurred with the formation of some defect. This phenomenon of small crystals physically embedded in larger crystals could only degrade the overall diffraction and materials properties of macromolecular crystals.

Mechanisms of growth for protein and virus crystals.

The growth of six protein and virus crystals was investigated in situ using atomic force microscopy. Most of the crystals grew principally on steps generated by two-dimensional nucleation on surfaces though some grew by development of spiral dislocations. Apoferritin grew by a rarely encountered mechanism, normal growth, usually associated only with melt or vapour phase crystallization. Cubic crystals of satellite tobacco mosaic virus (STMV) grew, at moderate to high levels of supersaturation, by the direct addition of three-dimensional nuclei followed by their rapid normal growth and lateral expansion, a mechanism not previously described to promote controlled and reproducible crystal growth from solutions. Biological macromolecules apparently utilize a more diverse range of growth mechanisms in their crystallization than any previously studied materials.

Light-scattering investigations of nucleation processes and kinetics of crystallization in macromolecular systems.

Quasi-elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, sigma = ln(c/s) where c and s are concentration and solubility of protein, varied from approximately 0.1 for pumpkin seed globulin to approximately 0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation-dependent critical nuclear size, R(c), for different macromolecular systems was estimated from time-dependent size-distribution analyses to be in the range of approximately 10(3) for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interfacial free energy was deduced to be 3.3-9.2 kJ mol(-1). These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3-50 kJ mol(-1). Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubility versus solution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early stages of the aggregation process.