Biomedical subjects

T S Baker

Publications and source records attributed to T S Baker.

At least 37 records · Page 2Linked to original sources

PubMed1998

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution.

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.

Amino Acid Sequence↗

PubMed1998

Assembly of a tailed bacterial virus and its genome release studied in three dimensions.

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Bacillus Phages↗

PubMed1998

Internal/structures containing transcriptase-related proteins in top component particles of mammalian orthoreovirus.

The structure of mammalian orthoreovirus top component particles, which are profoundly deficient in the content of double-stranded RNA genome, was determined at 30 A resolution by transmission cryoelectron microscopy and three-dimensional image reconstruction. Previously undetected, ordered densities, appearing primarily as pentameric flowers in the reconstruction, were seen to extend 65 A inwardly from the inner capsid at the icosahedral fivefold axes. Identically positioned but lower density elements were observed in two types of partially uncoated top component particles obtained by limited proteolysis. The levels of three inner-capsid proteins-lamda 1, lamda 3, and mu 2-were reduced in concert with the internal densities during proteolytic uncoating. Since lamda 3 contains the catalytic regions of the viral RNA polymerase and since both lamda 1 and mu 2 appear to play roles in transcription or mRNA capping, the internal structures are concluded to be complexes of the viral transcriptase-related enzymes. The findings have implications for the mechanisms of transcription and mRNA capping by orthoreovirus particles.

Animals↗

PubMed1998

Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryoelectron microscopy and image reconstruction.

Cryoelectron microscopy and three-dimensional image reconstruction analysis has been used to determine the structure of native and in vitro assembled cowpea chlorotic mottle virus (CCMV) virions and capsids to 25-A resolution. Purified CCMV coat protein was used in conjunction with in vitro transcribed viral RNAs to assemble RNA 1 only, RNA 2 only, RNA 3/4 only, and empty (RNA lacking) virions. The image reconstructions demonstrate that the in vitro assembled CCMV virions are morphologically indistinguishable from native virions purified from infected plants. The viral RNA (vRNA) is packaged similarly within the different types of virions. The centers of all assembled particles are generally devoid of density and the vRNA packs against the interior surface of the virion shell. The vRNA appears to adopt an ordered conformation at each of the quasi-threefold axes.

Bromovirus↗

PubMed1998

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Amino Acid Sequence↗

PubMed1998

Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01.

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of 111In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg 111In-labelled hCTM01 (100 M Bq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P < 0.0001) and 5.9 times higher than in blood (P < 0.0001). 111In activity in liver tissue was comparable to 111In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer.

Adult↗

PubMed1998

Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes.

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.

Amino Acid Sequence↗

PubMed1997

The structure of cucumber mosaic virus: cryoelectron microscopy, X-ray crystallography, and sequence analysis.

The three-dimensional structure of cucumber mosaic virus (CMV) was analyzed at 23 A resolution by cryoelectron microscopy and image reconstruction, demonstrating structural similarity to cowpea chlorotic mottle virus (CCMV), another member of the Bromoviridae family. The CMV structure was determined at 8 A resolution by X-ray crystallography with phases determined by single isomorphous replacement and refined by fivefold noncrystallographic symmetry averaging. The X-ray structure agreed with the electron microscopy reconstruction; the electron density is consistent with beta-barrel subunits arranged with T = 3 quasi-symmetry in an orientation similar to that observed in CCMV. Strong density surrounding the icosahedral threefold axes (quasi sixfold axes in the T = 3 particle) between 80 and 100 A from the particle center formed a cylinder of radius 11 A, similar to the density observed in the same region of CCMV. This density corresponds to the beta-annulus of CCMV, which differentiates hexamers from pentamers and determines the formation of the T = 3 particles. The CMV and CCMV amino acid sequences were aligned, providing information (based on the CCMV atomic model) about the probable distribution of residues in the three-dimensional structure of CMV.

Amino Acid Sequence↗

PubMed1997

On the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE2) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP.E3). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E2 scaffold. However, this investigation shows that the 12 large openings in the tE2 core permit the entrance of tBP, BP, and BP.E3 into a large central cavity where the BP component apparently binds near the tip of the tE2 trimer. The bone-shaped E3 molecule is anchored inside the central cavity through its interaction with BP. One end of E3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP.E3. Thus, E2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E3.BP components are determined by the geometric constraints of the underlying scaffold.

Freeze Fracturing↗

PubMed1997

A method for establishing the handedness of biological macromolecules.

When biological macromolecules are imaged in the transmission electron microscope (TEM), their inherent handedness is lost because the three-dimensional (3D) structure is projected onto a two-dimensional (2D) plane, and identical 2D projections can be made from either 3D enantiomer. Nevertheless, tilt experiments in the TEM can be used to determine handedness. These experiments have been performed successfully on negatively stained specimens. More recently, the method was applied to unstained, frozen-hydrated specimens imaged by means of cryoelectron microscopy (cryoTEM) methods. Tilt experiments involve recording two micrographs of the same particles at different tilt angles, computing enantiomeric reconstructions from particle images in one micrograph, predicting orientations of corresponding particles in the second micrograph, and comparing model projections with particle images in the second micrograph. In principle, this procedure can be used to determine the handedness of any biological macromolecule imaged by cryoTEM, provided the enantiomeric reconstructions are distinguishable.

Freezing↗

PubMed1997

Identification of spherical virus particles in digitized images of entire electron micrographs.

New methods are described that should facilitate high-resolution (5-10 A) image reconstructions from low-dose, low-contrast electron micrographs of frozen-hydrated specimens and processing of large, digital images produced by new imaging devices and modern electron microscopes. Existing techniques for automatic selection of images of individual biological macromolecules from electron micrographs are inefficient or unreliable. We describe the Crosspoint method (CP), which produces good quality solutions with relatively small miss rates and few false hits, and an extension of this method along with a procedure for refining its solution. Two algorithms for processing large images, one based on image subsampling, the other on image decomposition, are described. A large image is first compressed (e.g., by subsampling) and the CP method is applied to the compressed image to produce an initial solution. The information gathered at this stage is used to cut the original image into subimages and then to refine the particle coordinates in each subimage. An interactive environment for experimenting with particle identification methods is described.

Algorithms↗

PubMed1997

IRIS explorer software for radial-depth cueing reovirus particles and other macromolecular structures determined by cryoelectron microscopy and image reconstruction.

Structures of biological macromolecules determined by transmission cryoelectron microscopy (cryo-TEM) and three-dimensional image reconstruction are often displayed as surface-shaded representations with depth cueing along the viewed direction (Z cueing). Depth cueing to indicate distance from the center of virus particles (radial-depth cueing, or R cueing) has also been used. We have found that a style of R cueing in which color is applied in smooth or discontinuous gradients using the IRIS Explorer software is an informative technique for displaying the structures of virus particles solved by cryo-TEM and image reconstruction. To develop and test these methods, we used existing cryo-TEM reconstructions of mammalian reovirus particles. The newly applied visualization techniques allowed us to discern several new structural features, including sites in the inner capsid through which the viral mRNAs may be extruded after they are synthesized by the reovirus transcriptase complexes. To demonstrate the broad utility of the methods, we also applied them to cryo-TEM reconstructions of human rhinovirus, native and swollen forms of cowpea chlorotic mottle virus, truncated core of pyruvate dehydrogenase complex from Saccharomyces cerevisiae, and flagellar filament of Salmonella typhimurium. We conclude that R cueing with color gradients is a useful tool for displaying virus particles and other macromolecules analyzed by cryo-TEM and image reconstruction.

Animals↗

PubMed1997

The structure of alfalfa mosaic virus capsid protein assembled as a T=1 icosahedral particle at 4.0-A resolution.

K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.

Alfalfa mosaic virus↗

PubMed1997

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.

Chymotrypsin↗

PubMed1996

Biodistribution of (111)indium-labeled engineered human antibody CTMO1 in ovarian cancer patients: influence of protein dose.

Thirty-one patients suspected of having ovarian cancer received a single i.v. injection of radiolabeled (100 MBq (111)In) engineered human CTMO1 (hCTMO1) to investigate its potential as an internalizing drug carrier. hCTMO1 is a complementary-determining region-grafted human IgG4 monoclonal antibody recognizing an ovarian carcinoma-associated antigen, the MUC-1-gene product. The amount of radioactivity was determined in tumor tissue, various normal tissues, including liver biopsies, and blood samples obtained at laparotomy, 6 days after injection of either 0.1 or 1.0 mg hCTMO1/kg of body weight. Circulating antigen-15-3 was measurable in all patients before injection, and immune complex formation was already present at the end of infusion. In the 0.1 mg/kg group, most of the radioactivity was bound to immune complexes, whereas in the 1.0 mg/kg group, most was bound to IgG monomers. Increasing the hCTMO1 dose 10-fold did not influence the overall disappearance of (111)In from the blood, but the elimination half-life of (111)indium bound to immune complexes was increased 2-fold. Uptake in tumor tissue 6 days postinjection at the 0.1 mg/kg dose was 7.6 times higher (P = 0.0009) than in normal tissue and 2.5 times higher (P = 0.03) than in blood. At the 1.0 mg/kg dose, the uptake in tumor tissue was 14.0 times higher (P = 0.0003) than in normal tissue and 8.1 times higher (P = 0.0007) than in blood. Liver activity was substantial (23.7 +/- 10.5 and 18.3 +/- 6.7% of the injected dose/kg for the 0.1 and 1.0 mg/kg dose group, respectively). These results are superior to those found with other clinically tested anti-MUC-1 gene product antibodies. hCTMO1 seems to be a suitable carrier for cytotoxic agents in ovarian carcinoma patients; the better uptake results and tumor-to-blood ratios are obtained at the higher dose of 1.0 mg hCTMO1/kg body weight.

Adult↗

PubMed1996

Neutralizing antibody to human rhinovirus 14 penetrates the receptor-binding canyon.

The three-dimensional structure of intact human rhinovirus 14 (HRV-14) complexed with Fab fragments (Fab17-IA) from a strongly neutralizing antibody that binds bivalently to the virion has been determined to 4.0 angstrom resolution by a combination of X-ray crystallography and cryo-electron microscopy. In contradiction to the most commonly held model of antibody-mediated neutralization, Fab17-IA does not induce a conformational change in the HRV-14 capsid. Instead, the paratope of the antibody undergoes a large conformational change to accommodate the epitope. Unlike any previously described antibody-antigen structure, the conserved framework region of the antibody makes extensive contact with the viral surface. Fab17-IA penetrates deep within the canyon in which the cellular receptor for HRV-14 binds. Hence, it is unlikely that viral quaternary structure evolves merely to evade immune recognition. Instead, the shape and position of the receptor-binding region on a virus probably dictates receptor binding and subsequent uncoating events and has little or no influence on concealing the virus from the immune system.

Antibodies, Monoclonal↗

PubMed1996

Cryo-electron microscopy studies of empty capsids of human parvovirus B19 complexed with its cellular receptor.

The three-dimensional structures of human parvovirus B19 VP2 capsids, alone and complexed with its cellular receptor, globoside, have been determined to 26 resolution. The B19 capsid structure, reconstructed from cryo-electron micrographs of vitrified specimens, has depressions on the icosahedral 2-fold and 3-fold axes, as well as a canyon-like region around the 5-fold axes. Similar results had previously been found in an 8 angstrom resolution map derived from x-ray diffraction data. Other parvoviral structures have a cylindrical channel along the 5-fold icosahedral axes, whereas density covers the 5-fold axes in B19. The glycolipid receptor molecules bind into the depressions on the 3-fold axes of the B19:globoside complex. A model of the tetrasaccharide component of globoside, organized as a trimeric fiber, fits well into the difference density representing the globoside receptor. Escape mutations to neutralizing antibodies map onto th capsid surface at regions immediately surrounding the globoside attachment sites. The proximity of the antigenic epitopes to the receptor site suggests that neutralization of virus infectivity is caused by preventing attachment of viruses to cells.

Capsid↗

PubMed1996

Conserved features in papillomavirus and polyomavirus capsids.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Animals↗