Supplementary Components4. microscope built with a Zernike stage plate, a slim carbon film having a central opening, put into the comparative back again focal aircraft of the target zoom lens1,2. It shifts the stage of the spread electrons by /2, analogous for an optical stage contrast microscope. This considerably enhances the low-frequency info, allowing for in-focus, high contrast imaging6-8 (Extended Data Fig. 1). Consequently, low-contrast features difficult to detect in conventional cryoET images can be more readily identified. Open in a separate window Extended Data Figure 1 ZPC improves contrast of cryoET images and reveals detailed structural features of Syn5 infected cells(a) A conventional EM image of a Syn5-infected WH8109 cell. (b) A ZPC image of the same cell as shown in (a) under the same imaging conditions. WH8109 cells were imaged before infection and 65-70 minutes post infection. Even at this late infection time, some cells seemed to be newly infected. We reconstructed 58 ZPC tomograms of WH8109 cells (Figs. 1a, ?,2;2; Supplementary Videos 1-4 and Methods). The cells range from 0.7 to 1 1.0m in diameter. Although the cell envelope and thylakoid membrane (Fig. 1a-b) are roughly concentric, the thylakoid membrane does not fully enclose the inner compartment of the cell, nor does it seem to directly interact with the cell membrane. This Fulvestrant kinase inhibitor differs from the organization seen in other cyanobacteria9,10. Cyanobacteria also contain carboxysomes, polyhedral compartments encapsulating enzymes for carbon fixation11,12. Each WH8109 cell has, on average, four or five carboxysomes, with diameters ranging from 920 to 1160? (Fig. 1c). Ribosomes are wide-spread and abundant, forming several intracellular patches which contain polyribosomes (Fig. 1d). Open up in another window Shape 1 Zernike stage contrast cryoET allows direct reputation of cellular the different parts of the Syn5-contaminated WH8109 cells(a) Section look at of the Syn5 contaminated cell at past due stage of disease with parts labelled, including ribosomes (R), thylakoid membranes (T), carboxysomes (C), and infecting phages (I). Section and 3D annotated look at of above mobile parts are demonstrated in (b) C (e). (b) Thylakoid Fulvestrant kinase inhibitor membrane (green). (c) Carboxysome (blue). (d) Ribosome (crimson). (e) An infecting Syn5 phage (reddish colored) positioned regular to the top of contaminated cell. Yellow – cell envelope; magenta – phage progeny. Sections (b) C (e), size pubs = 500?. Open up in another window Shape 2 Zernike stage comparison cryoET of WH8109 cells before and after disease with Syn5 phageSection (remaining) and annotated (correct) sights of (a) an uninfected cell and contaminated cells at (b) early, (c) intermediate and (d) past due stages of disease. Sections demonstrated are 54? slabs extracted from the center of the tomograms. Cellular Mouse monoclonal to LSD1/AOF2 parts and phages are colored and labelled within the annotated look at in (c). Phage progeny could be sectioned off into three types predicated on size, form and internal denseness: procapsid (yellowish); extended capsid (red) and DNA-containing capsid (magenta). Cyanophage Syn5 that infects WH8109 cells is really a short-tailed icosahedral phage with a distinctive horn appendage in the vertex opposing towards the tail13 (Prolonged Data Fig. 2). Preliminary segmentation in our tomograms of contaminated cells determined Syn5 particles for the cell surface area, floating within the extracellular moderate, and Syn5 progeny in the cell. Multiple complete Fulvestrant kinase inhibitor and bare phage contaminants have emerged mounted on the cell surface area. Injection of viral DNA occurs at multiple sites on the bacterial envelope and does not appear to be a coordinated process. Fig. 1e shows a tubular density extending from the phage tail through the periplasm to the cytoplasm (Supplementary video 4), similar to observations in other phage-infected bacteria14,15. As infection progresses, increasing numbers of Syn5 phage progeny are observed inside the cells. Late in infection, the cell membrane deforms and ruptures, releasing the phage progeny (Fig. 2). Open in a separate window Extended Data Figure 2 ZPC-cryoEM single particle images of biochemically purified mature Syn5 phageThe particles are shown with the tail Fulvestrant kinase inhibitor pointing down and the wavy horn pointing.