Cryo electron micrographs of Tetrahymena thermophila solubilized mitochondrial membrane complexes - Glacios data
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ABSTRACT:
SUBMITTER: James Anthony Letts
PROVIDER: EMPIAR-10882 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Structures of <i>Tetrahymena</i>'s respiratory chain reveal the diversity of eukaryotic core metabolism.
Zhou Long L Maldonado María M Padavannil Abhilash A Guo Fei F Letts James A JA
Science (New York, N.Y.) 20220331 6595
Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multisubunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate <i>Tetrahymena thermophila</i> (Tt). Using cryo-electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt complex I (Tt-CI) and Tt-CIII<sub>2</sub> (Tt-SC I+III<sub>2</sub>) and ...[more]
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| EMPIAR-10919 | biostudies-other
Project description:The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.
| S-EPMC2903591 | biostudies-literature
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| S-EPMC8742875 | biostudies-literature
Project description:BackgroundIdentification and selection of protein particles in cryo-electron micrographs is an important step in single particle analysis. In this study, we developed a deep learning-based particle picking network to automatically detect particle centers from cryoEM micrographs. This is a challenging task due to the nature of cryoEM data, having low signal-to-noise ratios with variable particle sizes, shapes, distributions, grayscale variations as well as other undesirable artifacts.ResultsWe propose a double convolutional neural network (CNN) cascade for automated detection of particles in cryo-electron micrographs. This approach, entitled Deep Regression Picker Network or "DRPnet", is simple but very effective in recognizing different particle sizes, shapes, distributions and grayscale patterns corresponding to 2D views of 3D particles. Particles are detected by the first network, a fully convolutional regression network (FCRN), which maps the particle image to a continuous distance map that acts like a probability density function of particle centers. Particles identified by FCRN are further refined to reduce false particle detections by the second classification CNN. DRPnet's first CNN pretrained with only a single cryoEM dataset can be used to detect particles from different datasets without retraining. Compared to RELION template-based autopicking, DRPnet results in better particle picking performance with drastically reduced user interactions and processing time. DRPnet also outperforms the state-of-the-art particle picking networks in terms of the supervised detection evaluation metrics recall, precision, and F-measure. To further highlight quality of the picked particle sets, we compute and present additional performance metrics assessing the resulting 3D reconstructions such as number of 2D class averages, efficiency/angular coverage, Rosenthal-Henderson plots and local/global 3D reconstruction resolution.ConclusionDRPnet shows greatly improved time-savings to generate an initial particle dataset compared to manual picking, followed by template-based autopicking. Compared to other networks, DRPnet has equivalent or better performance. DRPnet excels on cryoEM datasets that have low contrast or clumped particles. Evaluating other performance metrics, DRPnet is useful for higher resolution 3D reconstructions with decreased particle numbers or unknown symmetry, detecting particles with better angular orientation coverage.
| S-EPMC7869254 | biostudies-literature
Project description: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 ).
| S-EPMC6858545 | biostudies-literature
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| S-EPMC6503763 | biostudies-literature
Project description:Tetrahymena thermophila, a classic ciliate model organism, has been shown to possess tubular mitochondrial cristae and highly divergent electron transport chain involving four transmembrane protein complexes (I-IV). Here we report cryo-EM structures of its ~8 MDa megacomplex IV2 + (I + III2 + II)2, as well as a ~ 10.6 MDa megacomplex (IV2 + I + III2 + II)2 at lower resolution. In megacomplex IV2 + (I + III2 + II)2, each CIV2 protomer associates one copy of supercomplex I + III2 and one copy of CII, forming a half ring-shaped architecture that adapts to the membrane curvature of mitochondrial cristae. Megacomplex (IV2 + I + III2 + II)2 defines the relative position between neighbouring half rings and maintains the proximity between CIV2 and CIII2 cytochrome c binding sites. Our findings expand the current understanding of divergence in eukaryotic electron transport chain organization and how it is related to mitochondrial morphology.
| S-EPMC10227065 | biostudies-literature
Project description:In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The ciliate Tetrahymena thermophila is a unicellular organism with elaborate endolysosomal pathways. Curiously, Tetrahymena and related protozoa have lost HOPS. Tetrahymena encodes multiple paralogs of most CORVET subunits, assembled into six distinct hexameric complexes, including a distinct Vps8 subunit. Here we analyze the composition of these complexes immunoisolated by affinity capture using the Vps8-FLAG paralogs as bait.
2019-12-19 | PXD014895 | Pride
Project description:Multifunctional acyltransferases are able to catalyze the esterification of various acyl-acceptors with activated fatty acids. Here we describe the identification of four proteins from Tetrahymena thermophila that share certain properties with mammalian acyltransferases regarding their predicted transmembrane structure, their molecular mass and the typical acyltransferase motif. Expression of the Tetrahymena sequences results in production of triacylglycerols and wax esters in recombinant yeast when appropriate substrates are provided. The in vitro characterization shows, that these enzymes are capable of esterifying different acyl-acceptors including fatty alcohols, diols, diacylglycerols and isoprenols with acyl-CoA thioesters. Based on these catalytic activities and the sequence similarities of the Tetrahymena proteins with acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family members, we conclude that we identified a new group of DGAT2-related multifunctional acyltransferases from protozoan organisms.
| S-EPMC3311841 | biostudies-literature
Project description:The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates.
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