Achieving High-Quality Tomographic Data Collection Using the 200 kV Transmission Electron Microscope with Dual Condenser Lens System.

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Subtomogram analysis using the Volta phase plate.

Structures assisting radical movements revealed

Many biological processes are governed by large assemblies of protein molecules. However, it is often very difficult to determine the three-dimensional structures of these assemblies using experimental biophysical techniques. Hence there is a need to develop computational approaches to fill this gap. This article presents an ant colony optimization approach to predict the structure of large multi-component protein complexes. Starting from pair-wise docking predictions, a multi-graph consisting of vertices representing the component proteins and edges representing candidate interactions is constructed. This allows the assembly problem to be expressed in terms of searching for a minimum weight spanning tree. However, because the problem remains highly combinatorial, the search space cannot be enumerated exhaustively and therefore heuristic optimisation techniques must be used. The utility of the ant colony based approach is demonstrated by re-assembling known protein complexes from the Protein Data Bank. The algorithm is able to identify near-native solutions for five of the six cases tested. This demonstrates that the ant colony approach provides a useful way to deal with the highly combinatorial multi-component protein assembly problem.

Preparation of DNA Substrates and Functionalized Glass Surfaces for Correlative Nanomanipulation and Colocalization (NanoCOSM) of Single Molecules.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Background Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Methods Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. Results We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ∼15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. Discussion This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ~ 15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.

AlphaFold can predict the structure of single- and multiple-chain proteins with very high accuracy. However, the accuracy decreases with the number of chains, and the available GPU memory limits the size of protein complexes which can be predicted. Here we show that one can predict the structure of large complexes starting from predictions of subcomponents. We assemble 91 out of 175 complexes with 10–30 chains from predicted subcomponents using Monte Carlo tree search, with a median TM-score of 0.51. There are 30 highly accurate complexes (TM-score ≥0.8, 33% of complete assemblies). We create a scoring function, mpDockQ, that can distinguish if assemblies are complete and predict their accuracy. We find that complexes containing symmetry are accurately assembled, while asymmetrical complexes remain challenging. The method is freely available and accesible as a Colab notebook https://colab.research.google.com/github/patrickbryant1/MoLPC/blob/master/MoLPC.ipynb.

Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes