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The number of protein structures being determined by cryo-electron microscopy is growing at an explosive rate. The number of protein structures being determined by cryo-electron microscopy is growing at an explosive rate.

Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biological macromolecules. The technological development of transmission electron microscopes, detectors and automated procedures in combination with user-friendly image processing software and ever-increasing computational power have made cryo-EM a successful and expanding technology over the past decade 1 . At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. The direct visualization of atom positions is essential for understanding the mechanisms of protein-catalysed chemical reactions, and for studying how drugs bind to and interfere with the function of proteins 2 . Here we report a 1.25 Å-resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution 3 ). We can visualize individual atoms in a protein, see density for hydrogen atoms and image single-atom chemical modifications. Beyond the nominal improvement in resolution, we also achieve a substantial improvement in the quality of the cryo-EM density map, which is highly relevant for using cryo-EM in structure-based drug design. Advances in electron cryo-microscopy allow the structure of apoferritin to be determined at a resolution that enables the visualization of individual atoms.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1 , 2 . However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA A receptor homopentamer 3 . Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. Advances in electron cryo-microscopy hardware allow proteins to be studied at atomic resolution.

Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å 2 ) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 μm), thus making it suitable for studies of many biologically important structures. Cryo-electron microscopy is widely employed in structural biology and uses phase contrast imaging. Here, the authors employ electron ptychography, a quantitative phase retrieval method for high-contrast, low-dose phase imaging of cryo-state rotavirus and immature HIV-1 virus-like particles, and show that electron ptychography is more efficient for phase recovery than conventional phase contrast imaging.

We report a fully automated procedure for the optimization and interpretation of reconstructions from cryo-electron microscopy (cryo-EM) data, available in Phenix as phenix.map_to_model. We applied our approach to 476 datasets with resolution of 4.5 Å or better, including reconstructions of 47 ribosomes and 32 other protein–RNA complexes. The median fraction of residues in the deposited structures reproduced automatically was 71% for reconstructions determined at resolutions of 3 Å or better and 47% for those at resolutions worse than 3 Å.

The dramatic growth in the use of cryo-electron microscopy (cryo-EM) to generate high-resolution structures of macromolecular complexes has changed the landscape of structural biology. The majority of structures deposited in the Electron Microscopy Data Bank (EMDB) at higher than 4-Å resolution were collected on Titan Krios microscopes. Although the pipeline for single-particle data collection is becoming routine, there is much variation in how sessions are set up. Furthermore, when collection is under way, there are a range of approaches for efficiently moving and pre-processing these data. Here, we present a standard operating procedure for single-particle data collection with Thermo Fisher Scientific EPU software, using the two most common direct electron detectors (the Thermo Fisher Scientific Falcon 3 (F3EC) and the Gatan K2), as well as a strategy for structuring these data to enable efficient pre-processing and on-the-fly monitoring of data collection. This protocol takes 3–6 h to set up a typical automated data collection session.

Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries of the technique requires quantitative knowledge about the contribution of multiple factors. Here, we present an in-depth analysis and optimization of the main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify the effects of the Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. By using this information to carefully maximize the experimental performance, it is now possible to routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps enables the building of better atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines given here are not limited to GPCRs and can be applied directly to other small proteins. There is a need to optimise cryo-EM data acquisition approaches to improve the resolution of GPCR cryo-EM structures to better than 2.5 Å, in order to use them for structure-based drug design purposes. Here, the authors present a systematic analysis of the main cryo-EM experimental parameters using three GPCRs as test cases, which is also of interest for the cryo-EM structure determination of other small membrane proteins.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Single-particle cryo-electron microscopy has enabled the structures of large proteins to be elucidated. This Perspective discusses technological improvements in this technique, focusing particularly on the past decade and likely future developments. In single-particle cryo-electron microscopy (cryo-EM), molecules suspended in a thin aqueous layer are rapidly frozen and imaged at cryogenic temperature in the transmission electron microscope. From the random projection views, a three-dimensional image is reconstructed, enabling the structure of the molecule to be obtained. In this article I discuss technological progress over the past decade, which has, in my own field of study, culminated in the determination of ribosome structure at 2.5-Å resolution. I also discuss likely future improvements in methodology.

Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery. Micropatterning of cryo-EM grids enables controlled adhesion of mammalian cells for cryo-ET-based structural studies. This approach leads to reproducible cellular morphology and improves focused ion beam thinning of cells for in-cell structural analyses.