RESUMEN
Atmospheric-pressure plasmas have been widely applied for surface modification and biomedical treatment because of their ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used to generate hydrophilic surfaces and eliminate surface contaminants to embed specimens onto grids. In addition, plasma treatment is a prerequisite for negative-stain and Quantifoil grids, whose surfaces are coated with hydrophobic amorphous carbon. Although the conventional glow discharge system has been used successfully in this purpose, there has been no further effort to take an advantage from the recent progress in the plasma field. Here, we developed a nonthermal atmospheric plasma jet system as an alternative tool for treatment of surfaces. The low-temperature plasma is a nonequilibrium system that has been widely used in biomedical area. Unlike conventional glow discharge systems, the plasma jet system successfully cleans and introduces hydrophilicity on the grid surface in the ambient environment without a vacuum. Therefore, we anticipate that the plasma jet system will have numerous benefits, such as convenience and versatility, as well as having potential applications in surface modification for both negative-stain and cryo-EM grid treatment.
Asunto(s)
Microscopía por Crioelectrón , Frío , Microscopía por Crioelectrón/instrumentación , VacioRESUMEN
Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
Asunto(s)
Microscopía por Crioelectrón/métodos , Microfluídica/instrumentación , Microfluídica/métodos , Análisis por Matrices de Proteínas/métodos , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodos , Microscopía por Crioelectrón/instrumentación , Complejo de la Endopetidasa Proteasomal , Agua/químicaRESUMEN
Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Proteínas Fúngicas/química , Adsorción , Aire , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas/química , Electricidad Estática , Agua/químicaRESUMEN
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
Asunto(s)
Microscopía por Crioelectrón/métodos , Cicloparafinas/química , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/aislamiento & purificación , Polímeros/química , Microscopía por Crioelectrón/instrumentaciónRESUMEN
Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (â¼5-10â ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700â ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Microfluídica/métodos , Músculo Esquelético/metabolismo , Subfragmentos de Miosina/química , Manejo de Especímenes/métodos , Animales , ConejosRESUMEN
In cryo-electron microscopy (cryo-EM) data collection, locating a target object is error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation shows its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and in locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection. The proposed approach will advance high-throughput and accurate data collection of images and diffraction patterns with minimal human operation.
Asunto(s)
Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/instrumentación , Recolección de Datos/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Aprendizaje Automático , Algoritmos , Microscopía por Crioelectrón/instrumentación , Procesamiento de Imagen Asistido por Computador/instrumentaciónRESUMEN
A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.
Asunto(s)
Microscopía por Crioelectrón/métodos , Desnaturalización Proteica , Proteínas/química , Proteínas/ultraestructura , Manejo de Especímenes/métodos , Adsorción , Aire , Microscopía por Crioelectrón/instrumentación , Ferritinas/química , Ferritinas/ultraestructura , Reproducibilidad de los Resultados , Propiedades de Superficie , Agua/químicaRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Microscopía por Crioelectrón/instrumentación , Oro , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on global B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to reduce noise levels and obtain more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , ARN Polimerasas Dirigidas por ADN/ultraestructura , Aprendizaje Profundo , SARS-CoV-2/ultraestructura , Proteínas Virales/ultraestructuraRESUMEN
Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against transmembrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further studies to understand the enzyme coupling mechanism. Here, we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data, we suggest a new simple hypothetical coupling mechanism for the molecular machine.
Asunto(s)
Microscopía por Crioelectrón/métodos , Complejo I de Transporte de Electrón/química , Escherichia coli/química , Proteínas de la Membrana/química , Nanoestructuras/química , Microscopía por Crioelectrón/instrumentación , Metabolismo Energético , Membrana Dobles de Lípidos/química , Lípidos/química , Modelos Moleculares , Estructura Molecular , Conformación ProteicaRESUMEN
The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
Asunto(s)
Microscopía por Crioelectrón/métodos , Descubrimiento de Drogas/métodos , Nanopartículas/química , Proteínas/química , Microscopía por Crioelectrón/instrumentación , Cristalización , Electrones , Microscopía Electrónica de Transmisión/instrumentación , Microscopía Electrónica de Transmisión/métodos , Flujo de TrabajoRESUMEN
The increasing popularity and adoption rate of cryo-electron microscopy (cryo-EM) is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an 'easy' test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for the quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras-Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single- and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.
Asunto(s)
Microscopía por Crioelectrón , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Recolección de Datos , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
In this chapter, we present an overview of a standard protocol to achieve structure determination at high resolution by Single Particle Analysis cryogenic Electron Microscopy using apoferritin as a standard sample. The purified apoferritin is applied to a glow-discharged support and then flash frozen in liquid ethane. The prepared grids are loaded into the electron microscope and checked for particle spreading and ice thickness. The microscope alignments are performed and the data collection session is setup for an overnight data collection. The collected movies containing two-dimensional images of the apoferritin sample are then processed to obtain a high-resolution three-dimensional reconstruction.
Asunto(s)
Apoferritinas/química , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Manejo de Especímenes/métodos , Animales , Apoferritinas/ultraestructura , Equidae , Congelación , Imagenología Tridimensional , Flujo de TrabajoRESUMEN
Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.
Asunto(s)
Microscopía por Crioelectrón/métodos , Recolección de Datos/métodos , Ribosomas/ultraestructura , Microscopía por Crioelectrón/instrumentación , Exactitud de los Datos , Humanos , Imagenología Tridimensional/métodos , Biología Molecular/métodos , Ribosomas/química , Imagen Individual de MoléculaRESUMEN
Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu's method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.
Asunto(s)
Microscopía por Crioelectrón , Secciones por Congelación , Microscopía Fluorescente , Microtomía , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/ultraestructura , Línea Celular , Células Cultivadas , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Humanos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Microtomía/métodosRESUMEN
Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.
Asunto(s)
Carbono , Microscopía por Crioelectrón/instrumentación , Microscopía Electrónica de Transmisión/instrumentaciónRESUMEN
Cryogenic electron microscopy (cryo-EM) is an important tool for determining the molecular structure of proteins and protein assemblies, including helical assemblies such as amyloid fibrils. In reconstruction of amyloid fibril structures from cryo-EM images, an important early step is the selection of fibril locations. This fibril picking step is typically done by hand, a tedious process when thousands of images need to be analyzed. Here we present a computer program called FibrilFinder that identifies the locations and directions of fibril segments in cryo-EM images, by using the properties that the fibrils should be linear objects and have widths within a specified range. The program outputs the fibril locations in text files compatible with the RELION density reconstruction program. After RELION is used to extract the particle image boxes contained in the fibril segments identified by FibrilFinder, a second program called FibrilFixer removes boxes that contain more than one fibril, for instance because two fibrils cross each other. As concrete and realistic examples, we describe the application of the two programs to cryo-EM images of two different amyloid fibrils, namely 40-residue amyloid-ß fibrils derived from human brain tissue by seeded growth and fibrils formed by the C-terminal half of the low-complexity domain of the RNA-binding protein FUS. Both examples of amyloid fibrils can be picked from cryo-EM images using the same set of FibrilFinder and FibrilFixer parameters, showing that this software does not require re-optimization for each sample. A set of 1337 cryo-EM images was analyzed in 17 min with one multi-core computer. The new fibril picking software should enable the rapid analysis and comparison of more helical structures using cryo-EM, and perhaps serve as part of the greater automation of the entire structure determination process.
Asunto(s)
Amiloide/química , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Microscopía por Crioelectrón/instrumentación , Humanos , Fragmentos de Péptidos/química , Proteína FUS de Unión a ARN/química , Relación Señal-Ruido , Programas Informáticos , Factores de TiempoRESUMEN
We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía por Crioelectrón/instrumentación , Tomografía con Microscopio Electrónico/instrumentaciónRESUMEN
The preparation of extremely thin samples, which are required for high-resolution electron microscopy, poses extreme risk of damaging biological macromolecules due to interactions with the air-water interface. Although the rapid increase in the number of published structures initially gave little indication that this was a problem, the search for methods that substantially mitigate this hazard is now intensifying. The two main approaches under investigation are (a) immobilizing particles onto structure-friendly support films and (b) reducing the length of time during which such interactions may occur. While there is little possibility of outrunning diffusion to the interface, intentional passivation of the interface may slow the process of adsorption and denaturation. In addition, growing attention is being given to gaining more effective control of the thickness of the sample prior to vitrification.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Complejos Multiproteicos/química , Aire , Carbono/química , Difusión , Grafito/química , Lípidos/química , Complejos Multiproteicos/aislamiento & purificación , Desnaturalización Proteica , Manejo de Especímenes/métodos , Estreptavidina/química , AguaRESUMEN
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.