Anti-6xHIS Tag polyclonal antibody [Biotin]

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 angstrom. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the similar to 19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at similar to 35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.

His-tagged molecular species, a ferrocene derivative and Protein A, were immobilized on electrode surfaces (Au and graphite) through formation of a chelated complex in the presence of Cu2+ cations used as bridging units. The complex was cleaved and the attached molecules were released from the electrode surface by applying reductive potential to the electrodes resulting in Cu2+ reduction, thus decomposing the chelate complex. The molecule release process was followed by cyclic voltammetry in case of the ferrocene derivative. His-tagged Protein A was additionally labeled with a fluorescent tag and its release was followed by fluorescence measurements in the solution and by impedance spectroscopy at the electrode. The studied release of the His-tagged redox species and biomolecules was considered as a new generic approach to the signal-controlled molecule release applicable in various biotechnological and biomedical applications.