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Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages

Nature Protocols volume 9, pages 2630–2642 (2014)Cite this article

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Abstract

Advances in electron cryotomography have provided new opportunities to visualize the internal 3D structures of a bacterium. An electron microscope equipped with Zernike phase-contrast optics produces images with markedly increased contrast compared with images obtained by conventional electron microscopy. Here we describe a protocol to apply Zernike phase plate technology for acquiring electron tomographic tilt series of cyanophage-infected cyanobacterial cells embedded in ice, without staining or chemical fixation. We detail the procedures for aligning and assessing phase plates for data collection, and methods for obtaining 3D structures of cyanophage assembly intermediates in the host by subtomogram alignment, classification and averaging. Acquiring three or four tomographic tilt series takes ∼12 h on a JEM2200FS electron microscope. We expect this time requirement to decrease substantially as the technique matures. The time required for annotation and subtomogram averaging varies widely depending on the project goals and data volume.

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Figure 1: ZPC electron microscope setup and photographs of the phase plate airlock, blade, disc and phase plate hole.
Figure 2: Zernike phase plate on-plane condition.
Figure 3: Phase plate setup and microscope alignment for ZPC cryoET imaging.
Figure 4: Contrast improvement by Zernike phase plate using Syn5-infected WH8109 cells as an example specimen.
Figure 5: Comparison of ZPC images of WH8109 cells taken with good and charging phase plates, and from centered and off-center phase plates.
Figure 6: ZPC cryoET of a Syn5-infected WH8109 cell.
Figure 7: Flowchart of subtomogram classification and alignment.

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Acknowledgements

This research has been supported by US National Institutes of Health (NIH) grants (P41GM103832 and R01GM080139) and the Robert Welch Foundation (Q1242). We thank D. Raytcheva, C. Haase-Pettingell and J.A. King for the supplies of Syn5 phage and for much assistance in preparing the samples for the experiments; K. Nagayama for suppling the phase plates; J. Flanagan for computational software; R.H. Rochat for photographs and part of the illustration in Figure 1; X. Liu for programs for cut-on frequency correction; and M. Kawasaki for many helpful discussions.

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Authors and Affiliations

  1. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, Texas, USA

    Wei Dai, Caroline Fu, Htet A Khant, Steven J Ludtke, Michael F Schmid & Wah Chiu

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  1. Wei Dai
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  2. Caroline Fu
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Contributions

W.D. carried out the Synechococcus WH8109 cell culture and Syn5 infection experiments, collected all ZPC cryoET tilt series and performed the data processing and subtomogram alignment. W.D. and C.F. prepared all the grids for ZPC cryoET imaging. C.F. and H.A.K. established the ZPC illumination configuration and the airlock system for phase plate disc exchange. S.J.L. and M.F.S. developed subtomogram processing programs. W.D., M.F.S. and W.C. performed the data analysis and interpretation. All authors contributed to the preparation of this paper.

Corresponding author

Correspondence to Wah Chiu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ZPC cryoEM images of a Syn5-infected WH8109 cell taken from good and charging phase plates.

All four images are taken under the same conditions at 25,000x magnification and 1.3 e-/Å2 exposure. (a) Image from a good phase plate. (b–d) Images from phase plates suffering from various amounts of charging. Scale bar: 200 nm.

Supplementary Figure 2 Screenshot of PPCont program for phase plate mapping and evaluation.

The good phase plates are reported in green, the possibly usable ones in orange, and the charging or otherwise unusable ones in pink.

Supplementary Figure 3 Diagram of low-dose setup for ZPC cryoET and the relative positions of the phase plate, Photo mode, and Focus mode in Search view.

Supplementary Figure 4 Flowchart of symmetry axis search procedure.

A symmetry axis search algorithm is used to determine if individual phage progeny particles have icosahedral symmetry and to align the particles to their icosahedral symmetry axes if symmetry is identified (Step 45).

Supplementary information

Supplementary Figure 1

ZPC cryoEM images of a Syn5-infected WH8109 cell taken from good and charging phase plates. (PDF 9824 kb)

Supplementary Figure 2

Screenshot of PPCont program for phase plate mapping and evaluation. (PDF 2600 kb)

Supplementary Figure 3

Diagram of low-dose setup for ZPC cryoET and the relative positions of the phase plate, Photo mode, and Focus mode in Search view. (PDF 1880 kb)

Supplementary Figure 4

Flowchart of symmetry axis search procedure. (PDF 462 kb)

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Dai, W., Fu, C., Khant, H. et al. Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages. Nat Protoc 9, 2630–2642 (2014). https://doi.org/10.1038/nprot.2014.176

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