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(b) Fourier-shell correlation indicates a resolution of 4.4 Å based on the ‘gold-standard’ Fourier shell correlation (FSC)=0.143 criterion.

and can, in the case of h Prx3, even deliver a near-atomic resolution.

The major impediment in achieving sub-nanometre resolution cryo-EM reconstructions across the proteome is the intrinsically low signal-to-noise ratio obtainable with such unstained biological samples embedded in ice.

It is a remarkably simple device, comprising a heated film of continuous, amorphous carbon located at the back-focal plane of the objective lens (Supplementary Fig. The VPP introduces a phase shift in the unscattered beam relative to scattered beams, increasing the phase contrast, and thus facilitates the observation of weak phase objects with markedly improved contrast.

(c) Power spectrum of electron micrograph featuring continuous signal without contrast transfer function (CTF) oscillations across the frequency spectrum.

(d) Four representative class averages of h Prx3 featuring random orientations promoted by thick ice (scale bar, 10 nm).

2).(a) Electron micrograph of frozen-hydrated h Prx3 dodecamers taken without a phase plate at 2.4 μm underfocus.

In thick ice, toroids are only readily discernible at high defocus (scale bar, 100 nm).

However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles ( often represent well-behaved specimens that are exceptions rather than the norm.However, we were not able to obtain a reconstruction that is consistent with the structure of h Prx3, let alone well resolved, despite investing more effort in particle picking and processing.Both data sets were made available for readers that would like to compare the respective raw data.We sought to elucidate the conformation that h Prx3 assumes in solution, without the potential for structural changes that may occur during assembly to higher-order structures or crystal packing.Frozen-hydrated specimens of h Prx3 were prepared by relaxing the blot force of the automatic plunging device to increase ice thickness.

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