RESUMEN
The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.
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Citoesqueleto de Actina/genética , Adenosina Trifosfatasas/genética , Membrana Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteínas de Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/química , Adenosina Trifosfatasas/química , Membrana Celular/química , Citocinesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Endosomas/química , Endosomas/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ.
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Membranas Mitocondriales , Ubiquinona , Humanos , Ubiquinona/química , Membranas Mitocondriales/metabolismo , Proteínas Portadoras , LípidosRESUMEN
Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.
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GTP Fosfohidrolasas , Fusión de Membrana , Mitocondrias , Membranas Mitocondriales , Humanos , Biocatálisis , Cardiolipinas/química , Cardiolipinas/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Mitocondrias/química , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , Mutación , Dominios Proteicos , Multimerización de Proteína , Dinámicas MitocondrialesRESUMEN
C-terminal tailing is an ancient and conserved form of peptide synthesis that protects cells from incomplete and potentially toxic translation products. Filbeck et al. (2020) and Crowe-McAuliffe et al. (2020) use structural, genetic, and biochemical approaches to elucidate the mechanisms driving C-terminal tailing.
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Bacterias , Ribosomas , Control de CalidadRESUMEN
Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.
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Proteínas Portadoras/genética , Factor 4E Eucariótico de Iniciación/genética , Iniciación de la Cadena Peptídica Traduccional , Ribosomas/genética , Animales , Sistemas CRISPR-Cas/genética , Humanos , Ratones , Biosíntesis de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , Control de Calidad , ARN Mensajero/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.
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Aciltransferasas/metabolismo , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/ultraestructura , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , RatasRESUMEN
We present a genetic interaction map of pairwise measures including â¼40% of nonessential S. pombe genes. By comparing interaction maps for fission and budding yeast, we confirmed widespread conservation of genetic relationships within and between complexes and pathways. However, we identified an important subset of orthologous complexes that have undergone functional "repurposing": the evolution of divergent functions and partnerships. We validated three functional repurposing events in S. pombe and mammalian cells and discovered that (1) two lumenal sensors of misfolded ER proteins, the kinase/nuclease Ire1 and the glucosyltransferase Gpt1, act together to mount an ER stress response; (2) ESCRT factors regulate spindle-pole-body duplication; and (3) a membrane-protein phosphatase and kinase complex, the STRIPAK complex, bridges the cis-Golgi, the centrosome, and the outer nuclear membrane to direct mitotic progression. Each discovery opens new areas of inquiry and-together-have implications for model organism-based research and the evolution of genetic systems.
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Epistasis Genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Evolución Biológica , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Glicoproteínas de Membrana , Mitosis , Complejos Multiproteicos/metabolismo , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Huso Acromático , Respuesta de Proteína DesplegadaRESUMEN
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
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Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína que Contiene ValosinaRESUMEN
During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2-4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline-arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8-13 and chromatin organization14-17.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Anafase , Cromatina/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Microtúbulos/química , Microtúbulos/metabolismo , Membrana Nuclear/química , Huso Acromático/metabolismoRESUMEN
Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.
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Proteínas de la Membrana/metabolismo , Animales , Membrana Celular/química , Membrana Celular/ultraestructura , Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Estructura Terciaria de ProteínaRESUMEN
During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in nonneuronal cells filopodia dynamics decrease the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results suggest that F-BAR domains are functionally diverse and highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.
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Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Neurogénesis , Neuronas/citología , Animales , Movimiento Celular , Corteza Cerebral/citología , Corteza Cerebral/embriología , Proteínas Activadoras de GTPasa , Ratones , Seudópodos/metabolismoRESUMEN
Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.
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Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/ultraestructura , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/ultraestructura , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/ultraestructura , Regulación Alostérica , Sitios de Unión/genética , Microscopía por Crioelectrón , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/genética , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/ultraestructura , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Proteínas Mitocondriales/química , Modelos Moleculares , Mutación , Factores de Elongación de Péptidos/química , Fosforilación , Dominios Proteicos , Rotación , Relación Estructura-ActividadRESUMEN
Translation is the set of mechanisms by which ribosomes decode genetic messages as they synthesize polypeptides of a defined amino acid sequence. While the ribosome has been honed by evolution for high-fidelity translation, errors are inevitable. Aberrant mRNAs, mRNA structure, defective ribosomes, interactions between nascent proteins and the ribosomal exit tunnel, and insufficient cellular resources, including low tRNA levels, can lead to functionally irreversible stalls. Life thus depends on quality control mechanisms that detect, disassemble and recycle stalled translation intermediates. Ribosome-associated Quality Control (RQC) recognizes aberrant ribosome states and targets their potentially toxic polypeptides for degradation. Here we review recent advances in our understanding of RQC in bacteria, fungi, and metazoans. We focus in particular on an unusual modification made to the nascent chain known as a "CAT tail", or Carboxy-terminal Alanine and Threonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.
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Biosíntesis de Proteínas , Ribosomas/metabolismo , Animales , Bacterias/genética , Bacterias/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Evolución Molecular , HumanosRESUMEN
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the â¼30-kb RNA genome in the â¼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Fosforilación , ARN Viral/metabolismo , COVID-19/genética , Proteínas de la Nucleocápside/metabolismo , Ribonucleoproteínas/metabolismo , GenómicaRESUMEN
BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.
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Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/ultraestructura , Chlorocebus aethiops , Microscopía por Crioelectrón , Dinaminas/metabolismo , Proteínas de Unión a Ácidos Grasos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Liposomas/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Modelos Moleculares , Estructura Terciaria de Proteína , TransfecciónRESUMEN
Current understanding of the neural processes underlying human grasping suggests that grasp computations involve gradients of higher to lower level representations and, relatedly, visual to motor processes. However, it is unclear whether these processes evolve in a strictly canonical manner from higher to intermediate and to lower levels given that this knowledge importantly relies on functional imaging, which lacks temporal resolution. To examine grasping in fine temporal detail here we used multivariate EEG analysis. We asked participants to grasp objects while controlling the time at which crucial elements of grasp programs were specified. We first specified the orientation with which participants should grasp objects, and only after a delay we instructed participants about which effector to use to grasp, either the right or the left hand. We also asked participants to grasp with both hands because bimanual and left-hand grasping share intermediate-level grasp representations. We observed that grasp programs evolved in a canonical manner from visual representations, which were independent of effectors to motor representations that distinguished between effectors. However, we found that intermediate representations of effectors that partially distinguished between effectors arose after representations that distinguished among all effector types. Our results show that grasp computations do not proceed in a strictly hierarchically canonical fashion, highlighting the importance of the fine temporal resolution of EEG for a comprehensive understanding of human grasp control.SIGNIFICANCE STATEMENT A long-standing assumption of the grasp computations is that grasp representations progress from higher to lower level control in a regular, or canonical, fashion. Here, we combined EEG and multivariate pattern analysis to characterize the temporal dynamics of grasp representations while participants viewed objects and were subsequently cued to execute an unimanual or bimanual grasp. Interrogation of the temporal dynamics revealed that lower level effector representations emerged before intermediate levels of grasp representations, thereby suggesting a partially noncanonical progression from higher to lower and then to intermediate level grasp control.
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Fuerza de la Mano , Corteza Motora/fisiología , Tiempo de Reacción , Adolescente , Adulto , Electroencefalografía/métodos , Femenino , Lateralidad Funcional , Humanos , Masculino , Análisis MultivarianteRESUMEN
Hermosillo et al. (J Neurosci 31: 10019-10022, 2011) have suggested that action planning of hand movements impacts decisions about the temporal order judgments regarding vibrotactile stimulation of the hands. Specifically, these authors reported that the crossed-hand effect, a confusion about which hand is which when held in a crossed posture, gradually reverses some 320 ms before the arms begin to move from an uncrossed to a crossed posture or vice versa, such that the crossed-hand is reversed at the time of movement onset in anticipation of the movement's end position. However, to date, no other study has attempted to replicate this dynamic crossed-hand effect. Therefore, in the present study, we conducted four experiments to revisit the question whether preparing uncrossed-to-crossed or crossed-to-uncrossed movements affects the temporo-spatial perception of tactile stimulation of the hands. We used a temporal order judgement (TOJ) task at different time stages during action planning to test whether TOJs are more difficult with crossed than uncrossed hands ("static crossed-hand effect") and, crucially, whether planning to cross or uncross the hands shows the opposite pattern of difficulties ("dynamic crossed-hand effect"). As expected, our results confirmed the static crossed-hand effect. However, the dynamic crossed-hand effect could not be replicated. In addition, we observed that participants delayed their movements with late somatosensory stimulation from the TOJ task, even when the stimulations were meaningless, suggesting that the TOJ task resulted in cross-modal distractions. Whereas the current findings are not inconsistent with a contribution of motor signals to posture perception, they cast doubt on observations that motor signals impact state estimates well before movement onset.
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Mano , Percepción del Tacto , Mano/fisiología , Humanos , Postura/fisiología , Percepción Espacial/fisiología , Tacto/fisiología , Percepción del Tacto/fisiologíaRESUMEN
Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.
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Microscopía por Crioelectrón , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Nucleótidos/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Modelos Moleculares , Péptidos/metabolismo , Peptidil Transferasas/metabolismo , Reproducibilidad de los Resultados , Solventes , Electricidad EstáticaRESUMEN
Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.
Asunto(s)
Apoferritinas , Electrones , Animales , Microscopía por Crioelectrón/métodos , Recolección de Datos , RatonesRESUMEN
The frontoparietal networks underlying grasping movements have been extensively studied, especially using fMRI. Accordingly, whereas much is known about their cortical locus much less is known about the temporal dynamics of visuomotor transformations. Here, we show that multivariate EEG analysis allows for detailed insights into the time course of visual and visuomotor computations of precision grasps. Male and female human participants first previewed one of several objects and, upon its reappearance, reached to grasp it with the thumb and index finger along one of its two symmetry axes. Object shape classifiers reached transient accuracies of 70% at â¼105 ms, especially based on scalp sites over visual cortex, dropping to lower levels thereafter. Grasp orientation classifiers relied on a system of occipital-to-frontal electrodes. Their accuracy rose concurrently with shape classification but ramped up more gradually, and the slope of the classification curve predicted individual reaction times. Further, cross-temporal generalization revealed that dynamic shape representation involved early and late neural generators that reactivated one another. In contrast, grasp computations involved a chain of generators attaining a sustained state about 100 ms before movement onset. Our results reveal the progression of visual and visuomotor representations over the course of planning and executing grasp movements.SIGNIFICANCE STATEMENT Grasping an object requires the brain to perform visual-to-motor transformations of the object's properties. Although much of the neuroanatomic basis of visuomotor transformations has been uncovered, little is known about its time course. Here, we orthogonally manipulated object visual characteristics and grasp orientation, and used multivariate EEG analysis to reveal that visual and visuomotor computations follow similar time courses but display different properties and dynamics.