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. 2017 Sep 5;25(9):1436-1441.e2.
doi: 10.1016/j.str.2017.06.017. Epub 2017 Jul 27.

Refined Cryo-EM Structure of the T4 Tail Tube: Exploring the Lowest Dose Limit

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Refined Cryo-EM Structure of the T4 Tail Tube: Exploring the Lowest Dose Limit

Weili Zheng et al. Structure. .
Free PMC article


The bacteriophage T4 contractile tail (containing a tube and sheath) was the first biological assembly reconstructed in three dimensions by electron microscopy at a resolution of ∼35 Å in 1968. A single-particle reconstruction of the T4 baseplate was able to generate a 4.1 Å resolution map for the first two rings of the tube using the overall baseplate for alignment. We have now reconstructed the T4 tail tube at a resolution of 3.4 Å, more than a 1,000-fold increase in information content for the tube from 1968. We have used legacy software (Spider) to show that we can do better than the typical 2/3 Nyquist frequency. A reasonable map can be generated with only 1.5 electrons/Å2 using the higher dose images for alignment, but increasing the dose results in a better map, consistent with other reports that electron dose does not represent the main limitation on resolution in cryo-electron microscopy.

Keywords: R-type pyocin; bacteriophage; bacteriophage tail; contractile injection system; helical filaments.


Figure 1
Figure 1. Cryo-EM analysis of the tail tubes
(A) An electron micrograph (Taylor et al., 2016) shows the tubes (black arrows) attached to the baseplates (white arrows). (B) A power spectrum generated from 26,320 overlapping tube segments. The red arrow indicates the first meridional, at a spacing of ~ 1/(40 Å). The yellow arrow indicates the putative “first” layer line, at a spacing of ~ 1/(133 Å), while the blue arrow (at ~ 1/(8.4 Å) indicates the furthest layer line seen. The images have been multiplied by the CTF to boost the SNR, which is why the Thon rings are enhanced.
Figure 2
Figure 2. Dose limitation in determining the near-atomic resolution structure
(A) A small section of the map calculated from only the third frame (corresponding to 1.5 electrons/Å2). (B) The same area in a map created from a weighted-sum of the first 20 frames. The first two frames have been discarded, so the pre-irradiation in (A) is 3 electrons/Å2. A number of bulky sidechains are labelled, showing that they can be positioned quite well in both maps. See also Figure S2 and Figure S3.
Figure 3
Figure 3. Overall reconstruction of the T4 tail tube
(A) An outside view of the T4 tail tube cryo-EM map aligned with aomic model. Three subunits from one ring are shown in yellow, red and magenta. An α-helix (bottom) and a β-sheet (top) from the yellow subunit project out and interface with both the red subunit and subunits in adjacent rings. (B) A close-up view from the lumen shows how the four β-strands of the red subunit become part of a continuous β-sheet which lines the lumen. See also Figure S4.
Figure 4
Figure 4. Comparison between T4 tail tube and pyocin tube
(A) A subunit from the T4 tail tube (red) is superimposed upon the refined subunit from the R-type pyocin tube (green). A comparison between the molecular surfaces colored according to the electrostatic potential for the T4 tail tube (B,C) and the R-type pyocin tube (D,E). The outer surfaces of the tubes are shown in (B,D) while the surface lining the lumen is shown in (C,E). Negative potential is red, while positive is blue, with neutral white. As expected for a tube transporting DNA, the lumen of the T4 tail tube (C) is quite negative, which would serve to “lubricate” the walls so that DNA would not stick. The red color corresponds to a potential of -10 kT/e whereas the blue color corresponds to a potential of +10 kT/e. See also Figure S5 and Table S1.

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