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Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 µm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs.
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Gotículas Lipídicas , Proteínas , Gotículas Lipídicas/metabolismo , Proteínas/metabolismo , Lipídeos , Metabolismo dos LipídeosRESUMO
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 foil grids.
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INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of â¼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].
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Inteligência Artificial , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte Ativo do Núcleo Celular , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , ProteômicaRESUMO
The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
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Elétrons , Processamento de Imagem Assistida por Computador , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia Computadorizada por Raios XRESUMO
Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.
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Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , SARS-CoV-2 , Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares , Reprodutibilidade dos Testes , SoftwareRESUMO
The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes. We demonstrate that a major cause is the delocalization of high-resolution Fourier components outside the field of view of the camera. We also show that, to correctly characterize the phenomenon, it is important to make a correction for linear magnification anisotropy. Even when the anisotropy is quite small, it is present at all defocus values before circular averaging of the Thon rings, as is also true before merging data from particles in many orientations. Under the conditions used in this paper, which are typical of those used in single-particle electron cryomicroscopy, fading of the Thon rings due to source coherence is negligible. The principal conclusion is that much higher values of defocus can be used to record images than is currently thought to be possible, keeping in mind that the above-mentioned delocalization of Fourier components will ultimately become a limitation. This increased understanding should give electron microscopists the confidence to use higher amounts of defocus to allow, for example, better visibility of their particles and Ewald sphere correction.
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Carbono/química , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , AnisotropiaRESUMO
Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.
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Microscopia Crioeletrônica , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/ultraestrutura , Autofagia , Modelos Moleculares , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TomografiaRESUMO
Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
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Estresse Oxidativo , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , MutaçãoRESUMO
Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.
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Microscopia Crioeletrônica/métodos , Coleta de Dados , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Ensaios de Triagem em Larga Escala , SoftwareRESUMO
Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.
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Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestrutura , Elongação Traducional da Cadeia Peptídica , Mapas de Interação de Proteínas , Transcrição Gênica , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Bacteriano , Humanos , Mycoplasma pneumoniae/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ribossomos/metabolismo , TranscriptomaRESUMO
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
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Betacoronavirus/química , Simulação de Dinâmica Molecular , Glicoproteína da Espícula de Coronavírus/química , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Glicosilação , Humanos , Domínios Proteicos , Multimerização Proteica , SARS-CoV-2RESUMO
The thickness of an object will, at some point, exceed the depth of field of a transmission electron microscope; the value at which this occurs, depends on the resolution and the wavelength considered. An image is then no longer a true projection of the 3D structure. This effect will be expressed in the power spectrum. Here, we first demonstrate this phenomenon experimentally, using carbon foils of different thicknesses and working at 40, 60, 80 and 300 kV. Since we determined the thicknesses of the foils by tomography, we are also able to confirm experimentally that in the case of a thick object, the Thon ring pattern can be described as the sum of the power spectra originating from thin, independently scattering slices. Thus, a sinc function envelope is observed that attenuates the Thon rings' amplitudes, yielding "nodes" in the pattern at which the amplitudes are zero. These nodes move to lower spatial frequencies with decreasing acceleration voltages and increasing thicknesses. Conversely, the object thickness can be directly derived from node positions at a particular acceleration voltage. We validate our approach by applying it to frozen-hydrated bacteria with experimentally determined thicknesses. Our model will contribute to more reliably determining the defocus to be used with contrast transfer function correction for thicker objects and at lower acceleration voltages.
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Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-Å-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex.
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RNA Polimerase III/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fator de Transcrição TFIIIB/química , Fatores de Transcrição/química , Transcrição Gênica , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.
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Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Benchmarking , Microscopia Crioeletrônica/normas , Bases de Dados Factuais , Tomografia com Microscopia Eletrônica/normas , HIV-1/química , HIV-1/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Biologia Molecular/métodos , Biologia Molecular/normas , Vírion/química , Vírion/ultraestruturaRESUMO
p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
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Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genéticaRESUMO
The demand for high-throughput data collection in electron microscopy is increasing for applications in structural and cellular biology. Here we present a combination of software tools that enable automated acquisition guided by image analysis for a variety of transmission electron microscopy acquisition schemes. SerialEM controls microscopes and detectors and can trigger automated tasks at multiple positions with high flexibility. Py-EM interfaces with SerialEM to enact specimen-specific image-analysis pipelines that enable feedback microscopy. As example applications, we demonstrate dose reduction in cryo-electron microscopy experiments, fully automated acquisition of every cell in a plastic section and automated targeting on serial sections for 3D volume imaging across multiple grids.
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Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Microscopia Eletrônica de Transmissão/métodos , Software , Humanos , Microscopia Eletrônica de Transmissão/instrumentaçãoRESUMO
Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.
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Automação Laboratorial/métodos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures of Pol III preinitiation complexes, comprising the 17-subunit Pol III and the heterotrimeric transcription factor TFIIIB, bound to a natural promoter in different functional states. Electron cryo-microscopy reconstructions, varying from 3.7 Å to 5.5 Å resolution, include two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system.
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Microscopia Crioeletrônica , DNA/metabolismo , DNA/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA Polimerase III/ultraestrutura , Sítios de Ligação , Domínio Catalítico , DNA/química , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , RNA Polimerase III/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fator de Transcrição TFIIIB/química , Fator de Transcrição TFIIIB/metabolismo , Fator de Transcrição TFIIIB/ultraestrutura , Fatores de Transcrição TFII/química , Iniciação da Transcrição GenéticaRESUMO
This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.
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Microscopia Crioeletrônica , Pesquisa/organização & administração , Microscopia Crioeletrônica/instrumentação , Fluxo de TrabalhoRESUMO
The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.