Figure 8. Fibrous bundles observed by cryo-focused-ion-beam (cryo-FIB) and cryo-electron tomography (cryo-ET) in in E. coli cells expressing ice nucleation protein (INP).

(A) Ion-beam image of a thin lamella containing E. coli cells expressing INP obtained from cryo-FIB milling. (B) Zoomed-in view of a cryo-transmission electron microscopy (TEM) image of the lamella in (A). Boxes with dashed lines indicate areas where tilt series were collected. (C, D) Snapshots from 3D cryo-tomograms reconstructed from tilt series collected in the boxed regions in (B) showing striking fibrous bundles (yellow arrowheads). The E. coli cell envelopes are indicated with thick dash lines. (E) Further examples of the fibrous bundles produced by INP-expressing E. coli. Size markers in (A) is 10 µm, in (B) is 2 µm, and in (C), (D), and (E) are 100 nm, respectively.

Figure 8—figure supplement 1. E. coli expressing ice nucleation protein (INP) mutant lacking R-coils show no fibre clusters as observed in those cells overexpressing wild-type INP.

(A–D) Representative snapshots from 3D cryo-tomograms showing cytoplasmic and extracellular features of various E. coli cells overexpressing an INP mutant in which all but the C-terminal R-coil have been replaced by WO-coils. All four images are in the same scale and the scale bar represents 100 nm.

Figure 8—video 1. Tomogram of an E. coli cell expressing an ice nucleation protein (INP).

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The tomogram shows fibrous bundles in the cytoplasm of a cell recombinantly expressing the INP from Pseudomonas borealis (PbINP). Tilt angle range is ±48°. Scale bar indicates 100 nm.

Figure 8—video 2. Tomogram of an E. coli cell expressing an ice nucleation protein (INP).

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Another example of fibrous bundles seen upon expression of INP from Pseudomonas borealis (PbINP). Tilt angle range is ±48°. Scale bar indicates 100 nm.