Journal of Structural Biology

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy -- rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

The cell membrane, which separates the cell from the environment, plays a key role in signal transduction, energy conversion and substance transport. Although previous membrane models have successfully interpreted some functions of the cell membrane, no consensus has been reached for the lack of direct and in situ evidence. Here, we characterized the high-resolution 3D structure of 293T cell membranes in situ for the first time by cryo-electron tomography. Due to the excellent thickness of our cryo-samples, we could clearly observe membrane proteins with an average molecular weight of 100 kD. By analysing tomograms, we found that the total thickness of a 293T plasma membrane is approximately 20 nm and that there is a 4-nm lipid bilayer structure within the membrane. We observed that membrane proteins and protein complexes with a similar height (7-11 nm) are densely embedded in the ectoplasmic side of 293T plasma membranes, whereas membrane proteins aggregate to form islands with heights reaching dozens of nanometres on the cytoplasmic side. Additionally, we measured the average sizes of membrane proteins on the cytoplasmic side of 293T plasma membranes and found them to be approximately 7 nm in length and 4 nm in width. Moreover, if more precise structural information is obtained in future studies, we will identify the molecular interactions and detailed structures of membrane protein clusters that can be easily distinguished on a 293T cell membrane. Our work represents the first in situ structural characterization of a native somatic cell membrane with cryo-electron tomography and advances cell membrane structural studies from the model prediction stage to the real structure observation stage.

BackgroundElectron 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. MethodsHere, 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. ResultsWe 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 [A] 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. DiscussionThis 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.

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

Cryo-electron tomography (cryo-ET) is fast developing from a tool primarily used to investigate structures of individual macromolecular complexes in situ into a high-resolution probe for molecular processes within diverse functional contexts in intact cells. It is thus increasingly necessary that the data are analyzed and quantified as completely as possible. But annotating and structurally characterizing macromolecular complexes with a high degree of completeness is a significant challenge, especially for smaller molecular targets. In particular, it is difficult to avoid incomplete localizations of complexes, false identifications, or losses during computational classification. To address these issues, we assessed parameters in data processing, including the role of voxel size in template matching, the effects of Volta phase plate imaging on localization, classification, and map refinement, and the extent to which multi-particle-based refinement of tiltseries improves these data processing steps. Our analyses provide practical guidelines that help maximize completeness in cellular cryo-ET data; accurate description of the sample is crucial for visual proteomics experiments, and these optimizations help ensure that data annotation and analysis are comprehensive.

Cryo-electron tomography (cryo-ET) is widely used to explore the 3D density of biomacromolecules. However, the heavy noise and missing wedge effect prevent directly visualizing and analyzing the 3D reconstructions. Here, we introduced REST, a deep learning strategy-based method to establish the relationship between low-quality and high-quality density and transfer this knowledge to restore signals in cryo-ET. Experimental results on purified ribosome and recombinant nucleosome datasets showed that REST had outstanding performance in denoising and compensating the missing wedge. The application in dynamic nucleosome structures suggests that REST has the capability to reveal individual macromolecules which present different conformations without subtomogram averaging. Moreover, REST could greatly improve the reliability of particle picking. These advantages enable REST to be a powerful tool for the straightforward interpretation of target macromolecules by visual inspection of the density and of a broad range of other applications in cryo-ET, such as segmentation, particle picking, and subtomogram averaging.

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate for negative staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step both stains yield comparable images to uranyl formate. Subsequently, using {beta}-galactosidase, it is shown that both stains can also successfully be employed for single particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are non-radioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample grid preparation - thus directly after purification - accessible to a broader range of scientists.

Cellular cryo electron tomography explores tissue and cells in their unstained flash-frozen native state, revealing in situ the structure of macromolecules together with their local environment and interactions with partners, also known as molecular sociology. To obtain thin samples, cryo-FIB milling is nowadays the most popular method, with impressive successes. The alternative, cryo-ultramicrotomy, is often overlooked on account of poorly reproducible attachment of cryo-sections to their support, resulting in extremely low throughput. We optimized the workflow, focusing on section adhesion and their support grids. We thus increased vitreous sections cryo electron tomography throughput to equal that of thin film, with typically several tens to hundreds of cryo-tomograms per sample. This open the way to new advances in cellular cryo electron tomography, as the method is devoid of beam damage, can provide large surfaces and serial sections of any type of sample from cells to tissues. In addition, section thickness can be tuned down to 30-50 nm, which may be an advantage for imaging small molecular complexes, such as DNA and nucleosomes.

Bicontinuous membranes in cell organelles epitomise natures ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterised by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. In cell organelles, their structure sizes around 50-500 nm and fluid nature make Transmission Electron Microscopy (TEM) the analysis method of choice to decipher nanostructural features. Here we present a tool to identify bicontinuous structures from TEM sections by comparison to mathematical "nodal surface" models, including the hexagonal lonsdaleite geometry. Our approach, following pioneering work by Deng and Mieczkowski (1998), creates synthetic TEM images of known bicontinuous geometries for interactive structure identification. We apply the method to the inner membrane network in plant cell chloroplast precursors and achieve a robust identification of the bicontinuous diamond surface as the dominant geometry in several plant species. This represents an important step in understanding their as yet elusive structure-function relationship.

Surface ice contamination is a persistent challenge in cryo-electron tomography (cryo-ET) workflows, where it can obscure regions of interest and contribute to curtaining artefacts during focused ion beam (FIB) milling. We demonstrate using high-pressure frozen yeast cells that a sublimation step within the scanning electron microscope (SEM) chamber before lamella milling visually removes surface ice and reduces sample roughness without detectable devitrification. While sublimation has been widely applied in cryo-SEM and volume imaging, it is not common on cryo-ET samples due to concerns about devitrification. Using tomographic reconstructions, we show that controlled sublimation improves lamella quality by reducing surface roughness and minimizing curtaining without compromising sample vitrification. Furthermore, subtomogram averaging of the 80S ribosome confirmed lamellae quality are preserved after sublimation. This approach offers a practical refinement to existing cryo-ET preparation protocols, requiring no additional instrumentation or workflow modifications.

In cryo-electron microscopy (cryo-EM), imaging of biological specimens is restricted by the limited field of view and by sample thickness. Hard X-ray imaging, with its ability to penetrate samples several tens of micrometers thick, offers a complementary approach for high-resolution visualization. A major concern is whether cryo-preserved samples can withstand the handling conditions at synchrotron facilities without excessive icing, and whether the radiation exposure during X-ray imaging compromises specimen integrity, thereby hindering subsequent attempts to achieve high-resolution 3D reconstructions via cryo-EM. To evaluate this, we deposited apoferritin samples on a cryo-EM grid, exposed them to varied X-ray doses typical for X-ray tomography experiments at a synchrotron facility, and subsequently analysed the exposed particles by cryo-EM. Despite the apparent damage sustained throughout the experiment, the samples remained amenable to cryo-EM analysis, with structural details at a resolution of [~]4 [A] at the highest absorbed X-ray dose of 100 MGy. By comparison, a similar cryo-EM dataset of the apoferritin particles that were not exposed to X-rays but were mounted on the same cryo-EM grid, resulted in a 3D reconstruction at 3.17 [A] resolution. Thus, while radiation damage may limit the high-resolution information in specimens processed by cryo-X-ray tomography, the cryo-preserved biological material exposed to these high X-ray doses can be still used for subsequent cryo-EM workflows aiming to obtain structural biology insights at intermediate to high resolution. These findings lay the groundwork for an integrated imaging workflow that combines X-ray and cryo-EM techniques to enable multiscale analysis of thick vitrified biological specimens.

Transmission electron cryo-microscopy (cryo-EM) allows for obtaining 3D structural information by imaging macromolecules embedded in thin layers of amorphous ice. To obtain high-resolution structural information, samples need to be thin to minimize inelastic scattering which blurs images. During data collection sessions, time spent on finding areas on the cryo-EM grid with optimal ice thickness should be minimized as imaging time on high-end Transmission Electron Microscope TEM systems is costly. Recently, grids covered with thin gold films have become popular due to their stability and reduced beam-induced motion of the sample. Gold foil grids have substantially different densities between the gold foil and ice, effectively resulting in the loss of dynamic range between thin and thick regions of ice, making it challenging to find areas with suitable ice thickness efficiently during grid screening and thus increase expensive imaging time. Here, an energy filter-based plasmon imaging is presented as a fast and easy method for grid screening of the gold grids.

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom ([A]) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 [A] and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 [A], respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset ([≤]1,000 micrographs) was sufficient to generate a 3.3 [A] reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg+2. In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 [A] resolution map of apoferritin with local resolution extending to 2.6 [A]. The map clearly showed two positions of an aromatic side chain. We also verified the optimal imaging conditions depending on different electron microscope and camera combinations. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

Mitochondrial division is a fundamental biological process that is crucial to cellular functionality and vitality. The prevailing hypothesis of Drp1 regulation with the involvement of ER and cytoskeleton does not account for all the observations. Following up our previous study in HeLa cells which led to the new hypothesis of mitochondrial division by budding, we employed in-situ Cryo-Electron Tomography (Cryo-ET) to visualize mitochondrial budding in intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, supporting the new hypothesis. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, no rings, or ring-like structures, or ER wrapping is presented in the budding regions suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles, distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. We propose these interacting vesicles may have mitochondrial origins.

In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. As a result, cryo-imaging reports obtained from non-trivial specimens including mammalian brain tissues are scarce and their physiological relevance remains to be determined. Here, we benchmarked plunge freezing with a variety of cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 microns thick and across several brain regions while keeping the tissue functional. By utilizing the knock-in (KI) mouse model with fluorescent astrocytes we have performed targeted cryo-FIB/SEM volume-EM imaging as well as targeted high-resolution cryoET imaging. Prior to cryoET, we have successfully generated lamellae in a semi-automated fashion on both LMIS (liquid metal ion source)- and plasma-based cryo-FIB/SEM instrumentation thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and astrocytes processes and validated the physiological relevance of our outputs based on the morphology of the corresponding cellular and subcellular features. The pipeline utilizes common vitrification setups and can be potentially extended toward alternative tissue specimens. Ultimately, we expect our approach to become an important step towards democratization of physiologically relevant in situ cryo-imaging studies.

Cryo-electron microscopy (cryo-EM) has become an indispensable technique for determining three-dimensional structures of biological macromolecules. A critical aspect of achieving high-resolution cryo-EM reconstructions is accurately determining and correcting for the microscopes contrast transfer function (CTF). The CTF introduces defocus-dependent distortions during imaging; if not properly accounted for, the CTF can distort features in and limit the resolution of 3D reconstructions. For tilt-series data used in cryo-electron tomography (cryo-ET), CTF estimation becomes even more challenging due to the tilt of the specimen, which introduces a defocus gradient across the field of view, as well as the low dose and signal in individual tilt images. Here, we describe a simple algorithm to improve the accuracy of CTF estimation of tilted images by leveraging the tilt-series alignment parameters determined for tomographic reconstruction to explicitly account for the tilted specimen geometry. In brief, each tilt image is divided into patches, each of which are then stretched according to their defocus shift. These are then summed to provide a coherent power spectra at the tilt axis, which can then be used in standard CTF estimation algorithms. This uses all the data in each image to enhance the visibility of Thon rings, thereby improving high-resolution CTF estimation and subsequent enhancements in the resolution of subtomogram averages.

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur, which is part of our cisTEM software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant et al., 2015). However, Unblur cannot accommodate motion due to more localized sample bending and distortions. Here, we present Unbend, extending Unblur by incorporating local motion correction using a three-dimensional cubic spline model. The 3D spline model is constructed using cubic B-splines along the exposure time axis, and bicubic B-splines within movie frames. Unbend is integrated into our cisTEM software with a new local motion visualization panel within the cisTEM graphical user interface. We processed movie frames from various in-situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method, which operates independently of the motion correction process, to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3-8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to [~]300%. The total and Von Mises equivalent strain shows a deformation scale of less than 1% in most of the samples, confirming that our model induces minimal additional distortion. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.

Frontier Structural Biology methods are transitioning from analysis of reconstituted macromolecular complexes in vitro to imaging of macromolecular assemblies within the physiological confines of the cell. Preparation of samples for in situ cryoEM analysis requires FIB milling or ultramicrotome sectioning, laborious and technically challenging procedures that are low-throughput and require a high degree of technical skills. We have devised a simple approach for cryoEM of nuclear macromolecular complexes that preserves to a high degree their physiological environment while removing the need for thin sectioning of the sample. The method requires only the preparation of nuclear extracts without additional purification or enrichment steps. We applied the method to obtain a 2.3 [A] cryoEM structure of nucleosomes visualised directly in the nuclear lysate of human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/732463v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15f4785org.highwire.dtl.DTLVardef@506f84org.highwire.dtl.DTLVardef@c95ceaorg.highwire.dtl.DTLVardef@1f326da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single molecule localization microscopy offers nowadays resolution nearly down to the molecular level with specific molecular labelling, thereby being a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles equivalent to cryo-EM single-particle analysis. In this analysis, we present particle averaging of fluorescently labelled Nup96 in the Nuclear Pore Complex followed by data analysis to show that Nup96 occurs as a dimer with in total 32 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.