RESUMO
SARS-CoV-2 is the third known coronavirus (CoV) that has crossed the animal-human barrier in the last two decades. However, little structural information exists related to the close genetic species within the SARS-related coronaviruses. Here, we present three novel SARS-related CoV spike protein structures solved by single particle cryo-electron microscopy analysis derived from bat (bat SL-CoV WIV1) and civet (cCoV-SZ3, cCoV-007) hosts. We report complex glycan trees that decorate the glycoproteins and density for water molecules which facilitated modeling of the water molecule coordination networks within structurally important regions. We note structural conservation of the fatty acid binding pocket and presence of a linoleic acid molecule which are associated with stabilization of the receptor binding domains in the "down" conformation. Additionally, the N-terminal biliverdin binding pocket is occupied by a density in all the structures. Finally, we analyzed structural differences in a loop of the receptor binding motif between coronaviruses known to infect humans and the animal coronaviruses described in this study, which regulate binding to the human angiotensin converting enzyme 2 receptor. This study offers a structural framework to evaluate the close relatives of SARS-CoV-2, the ability to inform pandemic prevention, and aid in the development of pan-neutralizing treatments.
Assuntos
Quirópteros , Microscopia Crioeletrônica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , SARS-CoV-2/química , Animais , Humanos , Quirópteros/virologia , COVID-19/virologia , Sítios de Ligação , Betacoronavirus , Motivos de Aminoácidos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Modelos Moleculares , Ligação ProteicaRESUMO
Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
Assuntos
Proteínas de Bactérias , Reparo do DNA , Escherichia coli , Fatores de Transcrição , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In traditional non-flammable electrolytes a trade-off always exists between non-flammability and battery performance. Previous research focused on reducing free solvents and forming anion-derived solid-electrolyte interphase. However, the contribution of solvated anions in boosting the stability of electrolyte has been overlooked. Here, we resolve this via introducing anions into Li+ solvation sheaths using anions with similar Gutmann donor number (DN) to that of solvents. Taking trimethyl phosphate fire-retardant (DN=23.0â kcal mol-1 ) and NO3 - (DN=22.2â kcal mol-1 ) as an example, NO3 - is readily involved in the Li+ solvation sheath and reduces the polarity of solvent. This results in boosted stability of electrolyte against Li. The developed non-flammable electrolyte has low viscosity, high ionic conductivity and is low cost. The reversibility of Li-Cu cell was improved to 99.49 % and the lifespan of practical LMBs was extended by >100 %.
RESUMO
Manganese-based Prussian Blue, Na2-δ Mn[Fe(CN)6 ] (MnPB), is a good candidate for sodium-ion battery cathode materials due to its high capacity. However, it suffers from severe capacity decay during battery cycling due to the destabilizing Jahn-Teller distortions it undergoes as Mn2+ is oxidized to Mn3+ . Herein, the structure is stabilized by a thin epitaxial surface layer of nickel-based Prussian Blue (Na2-δ Ni[Fe(CN)6 ]). The one-pot synthesis relies on a chelating agent with an unequal affinity for Mn2+ and Ni2+ ions, which prevents Ni2+ from reacting until the Mn2+ is consumed. This is a new and simpler synthesis of core-shell materials, which usually needs several steps. The material has an electrochemical capacity of 93â mA h g-1 , of which it retains 96 % after 500 charge-discharge cycles (vs. 37 % for MnPB). Its rate capability is also remarkable: at 4â A g-1 (ca. 55â C) it can reversibly store 70â mA h g-1 , which is also reflected in its diffusion coefficient of ca. 10-8 â cm2 s-1 . The epitaxial outer layer appears to exert an anisotropic strain on the inner layer, preventing the Jahn-Teller distortions it normally undergoes during de-sodiation.
RESUMO
Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.
Assuntos
Astrócitos/ultraestrutura , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Transmissão por Filtração de Energia/instrumentação , Microscopia Eletrônica de Transmissão por Filtração de Energia/métodos , Razão Sinal-Ruído , Coloração e Rotulagem/métodos , Animais , Encéfalo/patologia , Células HeLa , Humanos , Camundongos Endogâmicos C57BLRESUMO
The Redß protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redß, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redß to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redß, a defining member of the Redß/RecT protein family.
Assuntos
Bacteriófago lambda , DNA de Cadeia Simples , Bacteriófago lambda/química , DNA Complementar/metabolismo , DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Oligonucleotídeos/metabolismoRESUMO
In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/ultraestrutura , Imageamento Tridimensional , Modelos Moleculares , Ligação Proteica , Elongação da Transcrição GenéticaRESUMO
ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, ß and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate 'half-up' state to a condensed 'down' state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory 'up' state.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Microscopia Crioeletrônica , Conformação Proteica , Subunidades Proteicas/químicaRESUMO
A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120-400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721-739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 microm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão/instrumentação , Microscopia Eletrônica de Transmissão/métodosRESUMO
Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.
RESUMO
A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.
Assuntos
Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Silício , Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Elétrons , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Procedimentos Analíticos em MicrochipRESUMO
The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.
Assuntos
Microscopia Eletrônica de Transmissão/instrumentação , Animais , Drosophila/virologia , Desenho de Equipamento , Microscopia Eletrônica de Transmissão/estatística & dados numéricos , Vírion/ultraestruturaRESUMO
Alignment of the individual images of a tilt series is a critical step in obtaining high-quality electron microscope reconstructions. We report on general methods for producing good alignments, and utilizing the alignment data in subsequent reconstruction steps. Our alignment techniques utilize bundle adjustment. Bundle adjustment is the simultaneous calculation of the position of distinguished markers in the object space and the transforms of these markers to their positions in the observed images, along the bundle of particle trajectories along which the object is projected to each EM image. Bundle adjustment techniques are general enough to encompass the computation of linear, projective or nonlinear transforms for backprojection, and can compensate for curvilinear trajectories through the object, sample warping, and optical aberration. We will also report on new reconstruction codes and describe our results using these codes.
Assuntos
Análise de Fourier , Microscopia Eletrônica/métodos , Animais , Linhagem Celular , Células Cultivadas , Drosophila/citologia , Drosophila/ultraestrutura , Fibroblastos/ultraestrutura , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Camundongos Knockout , Microscopia Eletrônica/estatística & dados numéricos , Músculo Esquelético/ultraestrutura , Nodaviridae/ultraestrutura , Pontos Quânticos , Software , Design de Software , Tomografia/métodos , Tomografia/estatística & dados numéricos , TransfecçãoRESUMO
We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.