Exploiting radiation damage to map proteins in nucleoprotein complexes: The internal structure of bacteriophage T7

Abstract

In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique (“bubblegram imaging”) by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 sec) intervals between exposures of 17 electrons/Å2 each, bubbling starts in the third exposure, with 1 – 4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.

Supramolecular Organization and siRNA Binding of Hyaluronic Acid-Coated Lipoplexes for Targeted Delivery to the CD44 Receptor

“…(45,46) Bubblegram imaging has been previously used for the structural localization of proteins in vitrified specimens and DNA virus internal structure investigation. (47)(48)(49) We report here for the first time bubble formation evidencing siRNA localization within the lipoplexes. The uniform localization of the bubbles confirms the distribution of siRNA molecules within the lipoplexes, intercalated with the lipid membranes.…”

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

“…It is plausible that eccentric condensates represent NC-vRNA complexes that failed to be incorporated into the mature core. We obtained supporting evidence for this hypothesis by means of a labeling technique called bubblegram imaging (52,53). Vitrified samples imaged in an electron microscope undergo progressive damage by electron irradi- According to the chi-square test, significant differences with P values of Ͻ0.00001 were found for comparisons between WT and BIB-2 and between WT and the V165A mutant.…”

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

“…Severe radiation damage results in a local release of gas molecules such as hydrogen, which can give rise to white "bubbles" in electron micrographs. DNA-protein complexes tend to bubble at lower electron doses than protein (28,29). Taking advantage of this phenomenon, we were able to locate mitochondrial nucleoids in tomograms of isolated bovine heart mitochondria ( Fig.…”