We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining ...high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
Cryo-electron microscopy (cryo-EM) has emerged as a powerful structure determination technique. Its most prolific branch is single particle analysis (SPA), a method being used in a growing number of ...laboratories worldwide to determine high-resolution protein structures. Cryo-electron tomography (cryo-ET) is another powerful approach that enables visualization of protein complexes in their native cellular environment. Despite the wide-ranging success of cryo-EM, there are many methodological aspects that could be improved. Those include sample preparation, sample screening, data acquisition, image processing, and structure validation. Future developments will increase the reliability and throughput of the technique and reduce the cost and skill level barrier for its adoption.
Cryo-EM SPA requires good samples and large image datasets to produce near-atomic 3D maps of isolated proteins.Sample preparation is the main bottleneck in both SPA and cryo-ET.The current cryo-EM grid plunging technique is primitive. There is an urgent need to develop more efficient and consistent methods.Sample screening can be optimized and automated to improve the efficiency and productivity of cryo-EM.Data acquisition speed will continue to improve with prospects for at least a twofold increase in the next few years.More comprehensive data validation criteria and performance metrics must be defined and adopted as standard by the community.
With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with ...the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of them is the size of the target structure. Here, we report the use of a Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 Å. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.
Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a ...small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.
Electron-event representation saves the spatial and temporal coordinates of every electron with ultimate accuracy. Through maximum signal extraction, it will improve the performance of electron ...cryo-microscopy.
Significance Biological electron cryomicroscopy is limited by the radiation sensitivity of the samples and the consequent need to minimize exposure to the beam. This, in turn, results in low-contrast ...images with a poor signal-to-noise ratio. The current practice to improve phase contrast by defocusing results in contrast transfer functions necessitating image restoration to provide interpretable data. Phase plates enable in-focus phase contrast, but the existing ones, including the thin film Zernike-type phase plate, suffer from severe limitations, such as a short usable life span, fringing artifacts, and problems in using them in automated data acquisition procedures. The Volta phase plate presented here solves those problems and has the potential to become a practical solution for in-focus phase contrast in transmission electron microscopy.
We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to ∼200 °C to prevent contamination and enable the Volta potential effect. The phase shift is created “on the fly” by the central diffraction beam eliminating the need for precise phase plate alignment. Images acquired with the Volta phase plate (VPP) show higher contrast and unlike Zernike phase plate images no fringing artifacts. Following installation into the microscope, the VPP has an initial settling time of about a week after which the phase shift behavior becomes stable. The VPP has a long service life and has been used for more than 6 mo without noticeable degradation in performance. The mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the VPP it is used in a constructive way. The exact physics and/or chemistry behind the process causing the Volta potential are not fully understood, but experimental evidence suggests that radiation-induced surface modification combined with a chemical equilibrium between the surface and residual gases in the vacuum play an important role.
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•Tilt series acquisition in less than 5 min per target.•Robust compensation of specimen shifts in x, y and z.•Applicability to new (single-tilt axis) and old (dual-tilt axis) ...microscope stages.•Sub-nanometer subtomogram average with data collected in <50 min.
The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared to single particle approaches. A recent study reported significant acceleration of data acquisition by a fast-incremental single-exposure (FISE) tilt series scheme.
Here we improved the method and evaluated its efficiency and performance. We show that (1) FISE combined with the latest generation of direct electron detectors speeds up collection considerably, (2) previous generation (pre-2017) double-tilt axis Titan Krios holders are also suitable for FISE data acquisition, (3) x, y and z-specimen shifts can be compensated for, and (4) FISE tilt series data can generate averages of sub-nanometer resolution.
These advances will allow for a widespread adoption of cryoET for high-throughput in situ studies and high-resolution structure determination across different biological research disciplines.
The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo–electron tomography (cryo-ET) to produce three-dimensional snapshots ...of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed the native structure and organization of the cytoplasmic translation machinery. Analysis of a large dynamic structure—the nuclear pore complex—revealed variations detectable at the level of individual complexes. Cryo-ET was used to visualize previously elusive structures, such as nucleosome chains and the filaments of the nuclear lamina, in situ. Elucidation of the lamina structure provides insight into its contribution to metazoan nuclear stiffness.
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B ...receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gα
βγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gα
. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.
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