Identifying the assembly pathway of cyanophage inside the marine bacterium using electron cryo-tomography

Abstract

Advances in electron cryo-tomography open up a new avenue to visualize the 3-D internal structure of a single bacterium before and after its infection by bacteriophages in its native environment, without using chemical fixatives, fluorescent dyes or negative stains. Such direct observation reveals the presence of assembly intermediates of the bacteriophage and thus allows us to map out the maturation pathway of the bacteriophage inside its host.

Advances in Structural Biology and the Application to Biological Filament Systems

“…Individual components within the tomogram (e.g., actin filaments, microtubules, or ribosomes) can be extracted and averaged to obtain medium‐to‐high resolution structures (sub‐tomogram averaging). Newly developed phase plates further increase image contrast, making it easier to identify cellular components within cells . In vitro cryoET structures have been determined at medium resolution (8–9 Å) for structures with high symmetry like GroEL and the HIV capsid in the intact virus .…”

Cellular structural biology as revealed by cryo-electron tomography

“…Cells up to 1 μm in thickness can be imaged by EM, as shown for Ostreococcus tauri, the smallest eukaryotic cell (Henderson et al, 2007); this has provided a unique insight into the ultrastructure and characteristics of the cells during different stages of their cell cycle. The newly available phase plate technology for EM provides increased image contrast and therefore, makes it easier to identify fine cellular components within thicker specimens in situ (Dai et al, 2013(Dai et al, , 2014. Phase plates are typically positioned in the back focal plane of the objective lens of the EM and shift the phase of the scattered electron beam such that low-frequency information is amplified.…”

“…Phase plates are typically positioned in the back focal plane of the objective lens of the EM and shift the phase of the scattered electron beam such that low-frequency information is amplified. This is the information that contributes to image contrast, and higher contrast makes it possible to detect structural features within the data and, therefore, allows for the direct observation and analysis of the internal 3D structure of cells (Asano et al, 2015;Fukuda et al, 2015;Engel et al, 2015a,b;Dai et al, 2014Dai et al, , 2013 (Fig. 4).…”

Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography

“…In this study Danev and Nagayama also showed that as a result of the better contrast less particles are needed for a 3D reconstruction when compared to conventional brightfield data collection relying on defocused images. In the last years a number of other groups have used carbon fim phase plates and shown excellent results in particular for tomography [48][49][50]. Murata and coworkers [48] found, however, that -depending on electron energy and necessary carbon film thickness for the 90° phase shift, a loss in high resolution signal beyond about 7Å is observed.…”