Macromolecular crystallization in the structural genomics era.
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Biopolymers↗
Biomedical subjects
Alexander McPherson
Publications and source records attributed to Alexander McPherson.
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.
Animals↗
The crystallographic structure of brome mosaic virus.
The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.
Binding Sites↗
Rapid visualization at high resolution of pathogens by atomic force microscopy: structural studies of herpes simplex virus-1.
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.
DNA, Viral↗
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.
3T3 Cells↗
The structure of tomato aspermy virus by X-ray crystallography.
The three-dimensional structure of tomato aspermy virus (TAV) has been solved by X-ray crystallography and refined to an R factor of 0.218 for 3.4-40 A data (effective resolution of 4A). Molecular replacement, using cucumber mosaic virus (Smith et al., 2000), provided phases for the initial maps used for model building. The coat protein of the 280 A diameter virion has the canonical "Swiss roll" beta-barrel topology with a distinctive amino-terminal alpha-helix directed into the interior of the virus where it interacts with encapsidated RNA. The N-terminal helices are joined to the beta-barrels of protein subunits by extended polypeptides of six amino acids, which serve as flexible hinges allowing movement of the helices in response to local RNA distribution. Segments of three nucleotides of partially disordered RNA interact with the capsid, primarily through arginine residues, at interfaces between A and B subunits. Side chains of cys64 and cys106 form the first disulfide observed in a cucumovirus, including a unique cysteine, 106, in a region otherwise conserved. A positive ion, putatively modeled as a Mg(+)ion, lies on the quasi-threefold axis surrounded by three quasi-symmetric glutamate 175 side chains.
Amino Acid Sequence↗
Identification of DNA and RNA from retroviruses using ribonuclease A.
Retroviruses, such as human immunodeficiency virus (HIV), can be disrupted with chemical agents and made to disgorge their encapsidated nucleic acid. The products can be visualized by atomic force microscopy (AFM). Retroviruses may contain both viral genomic RNA and reverse transcribed DNA produced prior to integration into the host cell genome. It is necessary to know which molecules are RNA and which are DNA in order to interpret the events that transpire during infection. DNA, when imaged by AFM, is generally between one and two nanometers in thickness, more regular in its contours, and it is relatively uniform in height over its entire length; RNA, on the other hand, is less than a nanometer in thickness within single stranded regions, but varies dramatically in height over its length due to the presence of secondary structural domains. These observations, however, are often not definitive. Nonetheless, we have been able to tell one from the other using AFM, by exposing the molecules, in buffer, to moderate concentrations of RNase A. Upon exposure to the enzyme, the DNA, which cannot be cleaved, becomes coated with the protein, and the nucleic acid-protein complex exhibits a height of about three times that of the native molecule, appearing as thick cords. RNA, however, is degraded by the single strand specific RNase A into short, stable, presumably double-stranded segments, reflecting its pattern of secondary structure. Using this approach, we obtained evidence that reverse transcription of RNA into DNA may occur within the retroviral capsid.
DNA, Viral↗