Mechanisms, kinetics, impurities and defects: consequences in macromolecular crystallization.

The nucleation and growth of protein, nucleic acid and virus crystals from solution are functions of underlying kinetic and thermodynamic parameters that govern the process, and these are all supersaturation-dependent. While the mechanisms of macromolecular crystal growth are essentially the same as for conventional crystals, the underlying parameters are vastly different, in some cases orders of magnitude lower, and this produces very different crystallization processes. Numerous physical features of macromolecular crystals are of serious interest to X-ray diffractionists; the resolution limit and mosaicity, for example, reflect the degree of molecular and lattice order. The defect structure of crystals has an impact on their response to flash-cooling, and terminal crystal size is dependent on impurity absorption and incorporation. The variety and extent of these issues are further unique to crystals of biological macromolecules. All of these features are amenable to study using atomic force microscopy, which provides direct images at the nanoscale level. Some of those images are presented here.

An atomic force microscopy investigation of cyanophage structure.

Marine viruses have only relatively recently come to the attention of molecular biologists, and the extraordinary diversity of potential host organisms suggests a new wealth of genetic and structural forms. A promising technology for characterizing and describing the viruses structurally is atomic force microscopy (AFM). We provide examples here of some of the different architectures and novel structural features that emerge from even a very limited investigation, one focused on cyanophages, viruses that infect cyanobacteria (blue-green algae). These were isolated by phage selection of viruses collected from California coastal waters. We present AFM images of tailed, spherical, filamentous, rod shaped viruses, and others of eccentric form. Among the tailed phages numerous myoviruses were observed, some having long tail fibers, some other none, and some having no visible baseplate. Syphoviruses and a podovirus were also seen. We also describe a unique structural features found on some tailed marine phages that appear to have no terrestrial homolog. These are long, 450 nm, complex helical tail fibers terminating in a unique pattern of 3+1 globular units made up of about 20 small proteins.

Atomic force microscopy in imaging of viruses and virus-infected cells.

Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.

Atomic force microscopy investigation of viruses.

Atomic force microscopy (AFM) has proven to be a valuable approach to delineate the architectures and detailed structural features of a wide variety of viruses. These have ranged from small plant satellite viruses of only 17 nm to the giant mimivirus of 750 nm diameter, and they have included diverse morphologies such as those represented by HIV, icosahedral particles, vaccinia, and bacteriophages. Because it is a surface technique, it provides images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resolution. By enzymatic and chemical dissection of virions, internal structures can be revealed, as well as DNA and RNA. The method is relatively rapid and can be carried out on both fixed and unfixed samples in either air or fluids, including culture media. It is nondestructive and even non-perturbing. It can be applied to individual isolated virus, as well as to infected cells. AFM is still in its early development and holds great promise for further investigation of biological systems at the nanometer scale.

Nano-fibers produced by viral infection of amoeba visualized by atomic force microscopy.

In the course of an atomic force microscopy investigation of mimivirus, we observed that disrupted virions released masses of fibers that were several hundreds of nanometers in length and that could not be explained as nucleic acid or polysaccharide. The fibers exhibited a strong 7 nm periodicity along their lengths. They existed singly, and also as ribbons, cables, and in multi stranded coils. In the aggregate structures, the periodic bands of the individual fibers aligned laterally to produce ribbons and other superstructures having a corresponding pattern of 7 nm periodic transverse bands. We have not observed such fibers in studies of other virus and cellular systems. The fibers are mechanically flexible and resistant to breakage. Occasionally fibers were associated with toroidal protein complexes, assumed to be processive enzyme complexes, apparently in the act of modifying the fibers.

The three-dimensional structure of Mimivirus.

Mimivirus, the prototypic member of the new family of Mimiviridae, is the largest virus known to date. Progress has been made recently in determining the three-dimensional structure of the 0.75-microm diameter virion using cryo-electron microscopy and atomic force microscopy. These showed that the virus is composed of an outer layer of dense fibers surrounding an icosahedrally shaped capsid and an internal membrane sac enveloping the genomic material of the virus. Additionally, a unique starfish-like structure at one of the fivefold vertices, required by the virus for infecting its host, has been defined in more detail.

Fibrillar oligomers nucleate the oligomerization of monomeric amyloid beta but do not seed fibril formation.

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid beta (Abeta) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 x g, rich in beta-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Abeta FOs do not seed the formation of thioflavin T-positive fibrils from Abeta monomers but instead seed the formation of FOs from Abeta monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

Structural studies of the giant mimivirus.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60 degrees . The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Angstroms.

Investigation by atomic force microscopy of the structure of Ty3 retrotransposon particles.

Ty3, a member of the Metaviridiae family of long-terminal-repeat retrotransposons found in Saccharomyces cerevisiae, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs) that undergo processing and reverse transcription. The Ty3 structural proteins, capsid and nucleocapsid, contain major homology and nucleocapsid motifs similar to retrovirus capsid and nucleocapsid proteins, but Ty3 lacks a matrix-like structural domain amino terminal to capsid. Mass spectrometry analysis of Ty3 Gag3 processing products defined an acetylated Ser residue as the amino terminus of Gag3/p34, p27, and CA/p24 species and supported a model where p34 and p27 occur in phosphorylated forms. Using atomic force microscopy, VLPs were imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3. Wild-type VLPs were found to have a broad range of diameters, but the majority, if not all of the particles, exhibited arrangements of capsomeres on their surfaces which were consistent with icosahedral symmetry. Wild-type particles were in the range of 25 to 52 nm in diameter, with particles in the 42- to 52-nm diameter range consistent with T=7 symmetry. Both classes of mutant VLPs fell into a narrower range of 44 to 53 nm in diameter and appeared to be consistent with T=7 icosahedral symmetry. The smaller particles in the wild-type population likely correspond to VLPs that have progressed to reverse transcription or later stages, which do not occur in the protease and reverse transcriptase mutants. Ty3 VLPs did not undergo major external rearrangements during proteolytic maturation.

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV).

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.