RESUMO
Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line-based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography.
Assuntos
Tomografia com Microscopia Eletrônica , Processamento de Imagem Assistida por Computador/métodos , SoftwareRESUMO
Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.
Assuntos
Demência Frontotemporal , Príons , Encéfalo/metabolismo , Demência Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Príons/metabolismoRESUMO
Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force.
Assuntos
Proteoma/genética , Proteômica/métodos , Análise de Célula Única/métodos , Relação Estrutura-Atividade , AlgoritmosRESUMO
Cryo electron tomography allows macromolecular complexes within vitrified, intact, thin cells or sections thereof to be visualized, and structural analysis to be performed in situ by averaging over multiple copies of the same molecules. Image processing for subtomogram averaging is specific and cumbersome, due to the large amount of data and its three dimensional nature and anisotropic resolution. Here, we streamline data processing for subtomogram averaging by introducing an archiving system, Dynamo Catalogue. This system manages tomographic data from multiple tomograms and allows visual feedback during all processing steps, including particle picking, extraction, alignment and classification. The file structure of a processing project file structure includes logfiles of performed operations, and can be backed up and shared between users. Command line commands, database queries and a set of GUIs give the user versatile control over the process. Here, we introduce a set of geometric tools that streamline particle picking from simple (filaments, spheres, tubes, vesicles) and complex geometries (arbitrary 2D surfaces, rare instances on proteins with geometric restrictions, and 2D and 3D crystals). Advanced functionality, such as manual alignment and subboxing, is useful when initial templates are generated for alignment and for project customization. Dynamo Catalogue is part of the open source package Dynamo and includes tools to ensure format compatibility with the subtomogram averaging functionalities of other packages, such as Jsubtomo, PyTom, PEET, EMAN2, XMIPP and Relion.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares/ultraestrutura , SoftwareRESUMO
We present a new software package called Focus that interfaces cryo-transmission electron microscopy (cryo-EM) data collection with computer image processing. Focus creates a user-friendly environment to import and manage data recorded by direct electron detectors and perform elemental image processing tasks in a high-throughput manner while new data is being acquired at the microscope. It provides the functionality required to remotely monitor the progress of data collection and data processing, which is essential now that automation in cryo-EM allows a steady flow of images of single particles, two-dimensional crystals, or electron tomography data to be recorded in overnight sessions. The rapid detection of any errors that may occur greatly increases the productivity of recording sessions at the electron microscope.
Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Software , Automação , Razão Sinal-Ruído , Interface Usuário-ComputadorRESUMO
In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.
Assuntos
Bacteriófago PRD1/genética , Genoma Viral/genética , Nanotubos/virologia , Proteínas da Cauda Viral/metabolismo , Integração Viral/genética , Bacteriófago PRD1/crescimento & desenvolvimento , Bacteriófago PRD1/metabolismo , Capsídeo/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , DNA Viral/genética , Microscopia Eletrônica , Salmonella typhimurium/virologia , Integração Viral/fisiologiaRESUMO
Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI(+)], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI(+)] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.
Assuntos
Padrões de Herança/genética , Modelos Moleculares , Príons/química , Conformação Proteica , Leveduras/genética , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Microscopia de FluorescênciaRESUMO
The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.
Assuntos
Desmossomos/ultraestrutura , Epiderme/ultraestrutura , Citoesqueleto/química , Citoesqueleto/metabolismo , Desmoplaquinas/química , Desmoplaquinas/metabolismo , Caderinas de Desmossomos/química , Caderinas de Desmossomos/metabolismo , Desmossomos/química , Desmossomos/metabolismo , Epiderme/química , Epiderme/metabolismo , Humanos , Modelos Moleculares , Placofilinas/química , Placofilinas/metabolismo , gama CateninaRESUMO
With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography. We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.
RESUMO
Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used the single particle cryo-electron microscopy method to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 Å resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag lattice structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10-12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.
Assuntos
HIV-2 , Vírion , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Humanos , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , HIV-2/química , Vírion/química , Montagem de VírusRESUMO
Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.
Assuntos
Proteínas de Transporte , Endossomos , Endossomos/metabolismo , Transporte Proteico , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismoRESUMO
Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic 'focal pairs', which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3 nm for cells thicker than 500 nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Simulação por Computador , Processamento de Imagem Assistida por Computador/métodosRESUMO
Dynamo is a new software package for subtomogram averaging of cryo Electron Tomography (cryo-ET) data with three main goals: first, Dynamo allows user-transparent adaptation to a variety of high-performance computing platforms such as GPUs or CPU clusters. Second, Dynamo implements user-friendliness through GUI interfaces and scripting resources. Third, Dynamo offers user-flexibility through a plugin API. Besides the alignment and averaging procedures, Dynamo includes native tools for visualization and analysis of results and data, as well as support for third party visualization software, such as Chimera UCSF or EMAN2. As a demonstration of these functionalities, we studied bacterial flagellar motors and showed automatically detected classes with absent and present C-rings. Subtomogram averaging is a common task in current cryo-ET pipelines, which requires extensive computational resources and follows a well-established workflow. However, due to the data diversity, many existing packages offer slight variations of the same algorithm to improve results. One of the main purposes behind Dynamo is to provide explicit tools to allow the user the insertion of custom designed procedures - or plugins - to replace or complement the native algorithms in the different steps of the processing pipeline for subtomogram averaging without the burden of handling parallelization. Custom scripts that implement new approaches devised by the user are integrated into the Dynamo data management system, so that they can be controlled by the GUI or the scripting capacities. Dynamo executables do not require licenses for third party commercial software. Sources, executables and documentation are freely distributed on http://www.dynamo-em.org.
Assuntos
Microscopia Crioeletrônica , Software , Metodologias Computacionais , Linguagens de ProgramaçãoRESUMO
We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.
Assuntos
Elétrons , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Teorema de Bayes , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.
RESUMO
The robust alignment of tilt-series collected for cryo-electron tomography in the absence of fiducial markers, is a problem that, especially for tilt-series of vitreous sections, still represents a significant challenge. Here we present a complete software package that implements a cross-correlation-based procedure that tracks similar image features that are present in several micrographs and explores them implicitly as substitutes for fiducials like gold beads and quantum dots. The added value compared to previous approaches, is that the algorithm explores a huge number of random positions, which are tracked on several micrographs, while being able to identify trace failures, using a cross-validation procedure based on the 3D marker model of the tilt-series. Furthermore, this method allows the reliable identification of areas which behave as a rigid body during the tilt-series and hence addresses specific difficulties for the alignment of vitreous sections, by correcting practical caveats. The resulting alignments can attain sub-pixel precision at the local level and is able to yield a substantial number of usable tilt-series (around 60%). In principle, the algorithm has the potential to run in a fully automated fashion, and could be used to align any tilt-series directly from the microscope. Finally, we have significantly improved the user interface and implemented the source code on the graphics processing unit (GPU) to accelerate the computations.
Assuntos
Gráficos por Computador/tendências , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , SoftwareRESUMO
Cryo-tomography and subtomogram averaging are increasingly popular techniques for structural determination of macromolecular complexes in situ. They have the potential to achieve high-resolution views of native complexes, together with the details of their location relative to interacting molecules. The subtomogram averaging (StA) pipelines are well-established, with current developments aiming to optimise each step by reducing manual intervention and user decisions, following similar trends in single-particle approaches that have dramatically increased their popularity. Here, we review the main steps of typical StA workflows. We focus on considerations arising from the fact that the objects of study are embedded within unique crowded environments, and we emphasise those steps where careful decisions need to be made by the user.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Historically, structural biology has been largely centered on in vitro approaches as the dominant technique to obtain indispensable high-resolution data. In situ structural biology is now poised to contribute with high-precision observations in a near-physiological context. Mass spectrometry, electron tomography, and fluorescence microscopy are opening up new opportunities for structural analysis, including the study of the protein machinery in living cells. The complementarity between studies is increasingly used to reveal biologically significant observations. Here we compare two complementary studies addressing the mechanisms of vesicle tethering with in vitro and in situ approaches. Cryoelectron microscopy and live-cell imaging assisted by anchoring platforms team up to explore elusive mechanisms of exocytosis, showing directions of future research.
Assuntos
Membrana Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Citoplasma/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Exocitose , Proteínas/química , Membrana Celular/metabolismo , Citoplasma/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas/metabolismoRESUMO
Cryo-electron tomography (cryo-ET) allows three-dimensional (3D) visualization of frozen-hydrated biological samples, such as protein complexes and cell organelles, in near-native environments at nanometer scale. Protein complexes that are present in multiple copies in a set of tomograms can be extracted, mutually aligned, and averaged to yield a signal-enhanced 3D structure up to sub-nanometer or even near-atomic resolution. This technique, called subtomogram averaging (StA), is powered by improvements in EM hardware and image processing software. Importantly, StA provides unique biological insights into the structure and function of cellular machinery in close-to-native contexts. In this chapter, we describe the principles and key steps of StA. We briefly cover sample preparation and data collection with an emphasis on image processing procedures related to tomographic reconstruction, subtomogram alignment, averaging, and classification. We conclude by summarizing current limitations and future directions of this technique with a focus on high-resolution StA.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , SoftwareRESUMO
Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.