Orientation determination by wavelets matching for 3D reconstruction of very noisy electron microscopic virus images

Background  

In order to perform a 3D reconstruction of electron microscopic images of viruses, it is necessary to determine the orientation (Euler angels) of the 2D projections of the virus. The projections containing high resolution information are usually very noisy. This paper proposes a new method, based on weighted-projection matching in wavelet space for virus orientation determination. In order to speed the retrieval of the best match between projections from a model and real virus particle, a hierarchical correlation matching method is also proposed.     

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.     

Construction and expression of a novel recombinant anaphylatoxin, C5a-N19, as a probe for the human C5a receptor

We have constructed a novel recombinant C5a anaphylatoxin (C5a-N19) containing a 19-residue amino-terminal extension peptide, using a plasmid vector which secretes the nascent polypeptide to the Escherichia coli periplasmic space. C5a-N19 was purified from cell lysates by immunoaffinity chromatography using a monoclonal antibody which recognizes a portion of the amino-terminal extension peptide. C5a-N19 was characterized as biologically indistinguishable from the unmodified recombinant anaphylatoxin for release of lysosomal enzymes from dibutyryl-cAMP-differentiated U937 cells. In contrast to unmodified C5a, which is not recognized by anti-C5a antibodies following binding to its cellular receptor, receptor-bound C5a-N19 is recognized by the monoclonal antibody directed against the amino-terminal extension sequence. Because the monoclonal antibody recognizes the C5a-receptor complex on cells, this methodology is useful in fluorescence sorting of C5a receptor-positive cells. A C5a receptor affinity column was constructed by saturating monoclonal antibody bound to agarose with C5a-N19. Digitonin-solubilized C5a receptor from dibutyryl-cAMP-induced U937 cells was adsorbed to the matrix and eluted by dissociation of the ligand-receptor complex from the antibody. Analysis by SDS-polyacrylamide gel electrophoresis revealed a unique protein band at 41K, consistent with the molecular weight predicted from cross-linking experiments when the contribution of C5a is subtracted. Development of this recombinant C5a derivative provides a useful probe previously unavailable for the C5a receptor molecule.     

Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase

A method for the assay of 2-oxoglutarate decarboxylating enzymes based on determination of the reaction product [1-14C]succinate after precipitation of remaining 2-oxo[5-14C]glutarate with 2,4-dinitrophenyl hydrazine is reported. It is particularly useful for the study of the 2-oxoglutarate-coupled dioxygenase prolyl 4-hydroxylase (EC 1.14.11.2); it is superior to previously described assay methods of this enzyme with respect to simplicity of the procedure, speed, cost, and radiochemical safety. The results are highly reproducible, the standard deviation of repeated measurements being about 2% of the mean. The commercially available 2-oxo[5-14C]glutarate used in this study contained approximately 3% of radioactivity coeluting with succinate in HPLC and 1.5% of an unidentified radioactive compound as impurities, which contributed to the background.     

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.