Biomedical subjects

T S Baker

Publications and source records attributed to T S Baker.

114 records · Page 7Linked to original sources

PubMedDate not supplied

Low-resolution density maps from atomic models: how stepping "back" can be a step "forward".

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15-40 A), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.

Algorithms↗

PubMedDate not supplied

Magnification calibration and the determination of spherical virus diameters using cryo-microscopy.

The diameters of several frozen-hydrated, spherical viruses were determined using polyoma virus as either an external or an internal calibration standard. The methods described provide a reproducible and accurate way to calibrate microscope magnification. The measured diameters are in excellent agreement with respective measurements previously reported for aqueous samples at room temperature using X-ray diffraction methods. These results indicate that the native morphology and dimensions of biological macromolecules are better preserved in frozen-hydrated samples when compared with more conventional electron microscopy techniques such as negative-staining, metal shadowing or thin-sectioning.

Calibration↗

PubMedDate not supplied

The three-dimensional structure of frozen-hydrated Nudaurelia capensis beta virus, a T = 4 insect virus.

The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density.

Animals↗

PubMedDate not supplied

The three-dimensional structure of frozen-hydrated bacteriophage phi X174.

The three-dimensional structure of bacteriophage phi X174 (phi X174) was determined to approximately 2.6 nm resolution from images of frozen-hydrated 114 S particles. The outer surface of phi X174 is characterized by several prominent features: (i) 12 mushroom-shaped caps (approximately 7.1 nm wide x 3.8 nm high) are situated at each of the vertices of the icosahedral virion and extend to a maximum radius of 16.8 nm; (ii) a "collar" of density surrounds the base of each apical cap; and (iii) 20 conical protrusions (approximately 2.3 nm high) lie along the three-fold symmetry axes. The caps have a pentagonal morphology composed of five globular "subunits" and appear to be loosely connected to the underlying capsid. The distribution of the four gene products present in virions (60 copies each of gpF, gpG, and gpJ, and 12 copies of gpH), and the single-stranded DNA (ssDNA) genome cannot be directly discerned in the reconstructed density map, although plausible assignments can be made on the basis of solvent-excluded volume estimates and previous biochemical data. Thus, gpG accounts for most of the mass in the caps; gpH, a presumed cap protein, cannot be identified in part due to the symmetry-averaging procedures, but may be partially located within the interior of the capsid; and gpF and gpJ make up the remainder of the capsid. The genome appears to be less densely packaged inside the capsid compared to many dsDNA viruses whose nucleic acid is arranged in a liquid-crystalline state.

Bacteriophage phi X 174↗

PubMedDate not supplied

Polyoma virus 'hexamer' tubes consist of paired pentamers.

The discovery that the 72 capsomeres of the icosahedrally symmetric polyoma virus capsid are all pentamers shows that the expected quasi-equivalent bonding specificity is not conserved in the assembly of this virus coat protein. Tubular particles produced by polyoma and other papovaviruses seem to be polymorphic aggregates of capsomeres that may arise through variation in switching of the bonding specificity. Electron micrographs of wide and narrow classes of tubes were analysed by Kiselev and Klug using optical diffraction and optical filtering methods. The wide type were called 'hexamer' tubes because they consist of approximately hexagonally arrayed capsomeres that were assumed to be hexamers, in accord with the quasi-equivalence theory of icosahedral virus particle construction. The narrow type were called 'pentamer' tubes because the capsomeres are arrayed in a particular 'pentagonal tesselation' which arises from the pairing of pentamers across 2-fold axes of the surface lattice. Our reexamination of negatively-stained polyoma virus tubes by digital image processing of low-irradiation electron micrographs shows that all tubes are assemblies of paired pentameric capsomeres. We report here that the packing arrangement of the pentamers in the hexamer tubes is simply related to the pentagonal tessellation respresenting the packing in the narrow pentamer tubes. In all the tube structures examined, at least one pairwise contact between neighbouring pentamers closely resembles the contact between the pentavalent and hexavalent capsomeres in the icosahedral capsid.

Microscopy, Electron↗

PubMedDate not supplied

Rapid method for the GC/MS confirmation of 11-nor-9-carboxy-delta 9-tetrahydrocannabinol in urine.

A rigorously tested simple extraction procedure for the GC/MS determination of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-acid) from urine is described. This assay provides sensitivity, specificity and reliability and is particularly well suited for the routine determination of large numbers of samples. The THC-acid is detected as its trimethylsilyl derivative using electron impact ionization GC/MS with an on column detection limit below 1 ng/mL. The assay gives excellent reproducibility and linearity using the trideuterated analog of THC-acid as the internal standard. The procedure was evaluated with over 1300 samples and provided inter- and intra-day precision of 10% and 6% standard deviation, respectively.

Dronabinol↗