RESUMO
Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.
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COVID-19 , Pandemias , Microscopia Crioeletrônica/métodos , Humanos , SARS-CoV-2 , Imagem Individual de MoléculaRESUMO
Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes.
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Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Espectrometria de Massas , Proteoma , Proteômica , Animais , Humanos , Modelos Moleculares , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
Neurite self-recognition and avoidance are fundamental properties of all nervous systems1. These processes facilitate dendritic arborization2,3, prevent formation of autapses4 and allow free interaction among non-self neurons1,2,4,5. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, ß- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities1,2,4-13. Avoidance is observed between neurons that express identical protocadherin repertoires2,5, and single-isoform differences are sufficient to prevent self-recognition10. Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers10,14-20. Although these interactions have previously been characterized in isolation15,17-20, structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains11.
Assuntos
Caderinas/metabolismo , Caderinas/ultraestrutura , Microscopia Crioeletrônica , Neurônios/química , Neurônios/metabolismo , Animais , Caderinas/química , Caderinas/genética , Cristalografia por Raios X , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Modelos Moleculares , Neurônios/ultraestrutura , Domínios Proteicos , Multimerização Proteica , ProtocaderinasRESUMO
This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.
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Coleta de DadosRESUMO
Cryo-electron microscopy is a popular method for the determination of protein structures; however, identifying a sufficient number of particles for analysis can take months of manual effort. Current computational approaches find many false positives and require ad hoc postprocessing, especially for unusually shaped particles. To address these shortcomings, we develop Topaz, an efficient and accurate particle-picking pipeline using neural networks trained with a general-purpose positive-unlabeled learning method. This framework enables particle detection models to be trained with few sparsely labeled particles and no labeled negatives. Topaz retrieves many more real particles than conventional picking methods while maintaining low false-positive rates, is capable of picking challenging unusually shaped proteins (for example, small, non-globular and asymmetric particles), produces more representative particle sets and does not require post hoc curation. We demonstrate the performance of Topaz on two difficult datasets and three conventional datasets. Topaz is modular, standalone, free and open source ( http://topaz.csail.mit.edu ).
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Microscopia Crioeletrônica/métodos , Redes Neurais de Computação , Processamento de Imagem Assistida por ComputadorRESUMO
Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air-water or substrate-water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air-water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.
Assuntos
Ar , Apoferritinas/ultraestrutura , Microscopia Crioeletrônica/métodos , Hemaglutininas/ultraestrutura , Receptor de Insulina/ultraestrutura , Água/química , HumanosRESUMO
Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.
Assuntos
Microscopia Crioeletrônica/métodos , Animais , Tomografia com Microscopia Eletrônica , Frutose-Bifosfato Aldolase/ultraestrutura , Glutamato Desidrogenase/ultraestrutura , Coelhos , Manejo de EspécimesRESUMO
The field of electron tomography has benefited greatly from manual and semi-automated approaches to marker-based tilt-series alignment that have allowed for the structural determination of multitudes of in situ cellular structures as well as macromolecular structures of individual protein complexes. The emergence of complementary metal-oxide semiconductor detectors capable of detecting individual electrons has enabled the collection of low dose, high contrast images, opening the door for reliable correlation-based tilt-series alignment. Here we present a set of automated, correlation-based tilt-series alignment, contrast transfer function (CTF) correction, and reconstruction workflows for use in conjunction with the Appion/Leginon package that are primarily targeted at automating structure determination with cryogenic electron microscopy.
Assuntos
Microscopia Crioeletrônica/métodos , Dependovirus , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Automação Laboratorial/métodos , SoftwareRESUMO
As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.
Assuntos
Microscopia Crioeletrônica/métodos , Dependovirus , Interpretação de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Molecular/métodos , Fondaparinux , Substâncias Macromoleculares/análise , Polissacarídeos/análiseRESUMO
Compared to the field of X-ray crystallography, the field of single particle three-dimensional electron microscopy has few reliable metrics for assessing the quality of 3D reconstructions. New metrics are needed that can determine whether a given 3D reconstruction accurately reflects the structure of the particles from which it was derived or instead depicts a plausible though incorrect structure due to coarse misalignment of particles. Here an empirical procedure is presented for differentiating between a reconstruction with well-aligned particles and a reconstruction with grossly misclassified particles. For a given dataset, 3D reconstructions are computed from subsets of particles with decreasing numbers of particles contributing to the reconstruction. A plot of inverse resolution vs. the logarithm of the number of particles (a "ResLog" plot) provides metrics for the reliability of the reconstruction and the overall quality of the dataset and processing. Specifically, the y-intercept of a regression line provides a measure of the relative accuracy of the particle alignment and classification, and the slope is an indicator of the overall data quality including the imaging conditions and processing steps. ResLog plots can also be used to optimize conditions for data collection and reconstruction parameters. Although resolution estimates can vary by method of calculation, ResLog-derived parameters are consistent whether calculated by Fourier shell correlation or Fourier neighbor correlation, or a new coordinate-based metric that serves as a yardstick for structures where atomic coordinates are available. ResLog plots could become part of a standard set of parameters to be included in 3D reconstruction reports.
Assuntos
Microscopia Crioeletrônica/métodos , Algoritmos , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Microscopia Eletrônica/métodosRESUMO
Cryogenic-focused ion beam (cryo-FIB) instruments became essential for high-resolution imaging in cryo-preserved cells and tissues. Cryo-FIBs use accelerated ions to thin samples that would otherwise be too thick for cryo-electron microscopy (cryo-EM). This allows visualizing cellular ultrastructures in near-native frozen hydrated states. This review describes the current state-of-the-art capabilities of cryo-FIB technology and its applications in structural cell and tissue biology. We discuss recent advances in instrumentation, imaging modalities, automation, sample preparation protocols, and targeting techniques. We outline remaining challenges and future directions to make cryo-FIB more precise, enable higher throughput, and be widely accessible. Further improvements in targeting, efficiency, robust sample preparation, emerging ion sources, automation, and downstream electron tomography have the potential to reveal intricate molecular architectures across length scales inside cells and tissues. Cryo-FIB is poised to become an indispensable tool for preparing native biological systems in situ for high-resolution 3D structural analysis.
Assuntos
Microscopia Crioeletrônica , Microscopia Crioeletrônica/métodos , Humanos , Animais , Íons/químicaRESUMO
CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.
RESUMO
CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.
RESUMO
Over the past decade, cryo-electron microscopy (cryoEM) has emerged as an important method for determining near-native, near-atomic resolution 3D structures of biological macromolecules. To meet the increasing demand for cryoEM, automated methods that improve throughput and efficiency of microscope operation are needed. Currently, the targeting algorithms provided by most data-collection software require time-consuming manual tuning of parameters for each grid, and, in some cases, operators must select targets completely manually. However, the development of fully automated targeting algorithms is non-trivial, because images often have low signal-to-noise ratios and optimal targeting strategies depend on a range of experimental parameters and macromolecule behaviors that vary between projects and collection sessions. To address this, Ptolemy provides a pipeline to automate low- and medium-magnification targeting using a suite of purpose-built computer vision and machine-learning algorithms, including mixture models, convolutional neural networks and U-Nets. Learned models in this pipeline are trained on a large set of images from real-world cryoEM data-collection sessions, labeled with locations selected by human operators. These models accurately detect and classify regions of interest in low- and medium-magnification images, and generalize to unseen sessions, as well as to images collected on different microscopes at another facility. This open-source, modular pipeline can be integrated with existing microscope control software to enable automation of cryoEM data collection and can serve as a foundation for future cryoEM automation software.
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Algoritmos , Software , Humanos , Microscopia Crioeletrônica/métodos , Aprendizado de Máquina , Coleta de DadosRESUMO
Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ (inside cells) resembles the canonical structure determined in vitro 25 years ago. Nucleosome structure in situ is otherwise poorly understood. Using cryo-electron tomography (cryo-ET) and 3D classification analysis of budding yeast cells, here we find that canonical nucleosomes account for less than 10% of total nucleosomes expected in situ. In a strain in which H2A-GFP is the sole source of histone H2A, class averages that resemble canonical nucleosomes both with and without GFP densities are found ex vivo (in nuclear lysates), but not in situ. These data suggest that the budding yeast intranuclear environment favors multiple non-canonical nucleosome conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average budding yeast nucleosome's DNA is partially detached in situ.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Nucleossomos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina , Histonas/genética , Saccharomycetales/genéticaRESUMO
Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to ~10 min with Autoscreen compared to ~6 h using previous methods which are hampered by their inability to account for high variability between grids. This protocol first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12-grid automated screening session.
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Sistemas Computacionais , Gelo , Microscopia Crioeletrônica , Automação , AlgoritmosRESUMO
Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized for several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness and particle behavior. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel purpose-built image-processing and microscope-handling algorithms are incorporated into the automated data-collection software Leginon, to provide an open-source solution for fully automated high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen-exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid autoscreen session and conventional manual screening by 5 expert microscope operators are presented. On average, Autoscreen reduces operator time from â¼6â h to <10â min and provides a percentage of suitable images for evaluation comparable to the best operator. The ability of Smart Leginon to target holes that are particularly difficult to identify is analyzed. Finally, the utility of Smart Leginon is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. The fully automated functionality of Smart Leginon significantly reduces the burden on operator screening time, improves the throughput of screening and recovers idle microscope time, thereby improving availability of cryoEM services.
Assuntos
Processamento de Imagem Assistida por Computador , Software , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , ElétronsRESUMO
The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Anquirinas , Proteína 1 de Troca de Ânion do Eritrócito/análise , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , EspectrinaRESUMO
Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the "Waffle Method" protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100-200 nm and dimensions of approximately 12 µm width and 15-20 µm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h. Graphical abstract.
RESUMO
Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.