Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET.

[Display omitted] • We present a method that allows the cryo-EM/ET (electron cryo-microscopy/tomography) imaging of rod-shaped bacteria orthogonal to their longest axis, which is practically impossible using standard vitrification methods on cryo-EM grids. • We demonstrate straightforward manufacture of the honeycomb discs we introduce. • We present the first ever complete view of an FtsZ ring in E. coli using the method in 1). • We demonstrate that the honeycomb discs are superior in terms of mechanical and imaging performance, because the cells are surrounded by conducting gold metal. • We show that molecular resolutions can be obtained after subtomogram averaging from the FIB-milled honeycomb lamellae (ribosomes at 6.7 Å resolution). • We discuss how the honeycomb discs may have other important applications, for example to overcome preferred orientations of single particles in cryo-EM. Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations. [ABSTRACT FROM AUTHOR]