Bending-torsional elasticity and energetics of the plus-end microtubule tip

Microtubules (MTs), mesoscopic cellular filaments, grow primarily by the addition of GTP-bound tubulin dimers at their dynamic flaring plus-end tips. They operate as chemomechanical energy transducers with stochastic transitions to an astounding shortening motion upon hydrolyzing GTP to GDP. Time-resolved dynamics of the MT tip—a key determinant of this behavior—as a function of nucleotide state, internal lattice strain, and stabilizing lateral interactions have not been fully understood. Here we use atomistic simulations to study the spontaneous relaxation of complete GTP-MT and GDP-MT tip models from unfavorable straight to relaxed splayed conformations and to comprehensively characterize the elasticity of MT tips. Our simulations reveal the dominance of viscoelastic dynamics of MT protofilaments during the relaxation process, driven by the stored bending-torsional strain and counterbalanced by the interprotofilament interactions. We show that the posthydrolysis MT tip is exposed to higher activation energy barriers for straight lattice formation, which translates into its inability to elongate. Our study provides an information-driven Brownian ratchet mechanism for the elastic energy conversion and release by MT tips and offers insights into the mechanoenzymatics of MTs

Microtubule dynamics are defined by conformations and stability of clustered protofilaments

Microtubules are dynamic cytoskeletal polymers that add and lose tubulin dimers at their ends. Microtubule growth, shortening and transitions between them are linked to GTP hydrolysis. Recent evidence suggests that flexible tubulin protofilaments at microtubule ends adopt a variety of shapes, complicating structural analysis using conventional techniques. Therefore, the link between GTP hydrolysis, protofilament structure and microtubule polymerization state is poorly understood. Here, we investigate the conformational dynamics of microtubule ends using coarse-grained modeling supported by atomistic simulations and cryo-electron tomography. We show that individual bent protofilaments organize in clusters, transient precursors to a straight microtubule lattice, with GTP-bound ends showing elevated and more persistent cluster formation. Differences in the mechanical properties of GTP- and GDP-protofilaments result in differences in intra-cluster tension, determining both clustering propensity and protofilament length. We propose that conformational selection at microtubule ends favors long-lived clusters of short GTP-protofilaments that are more prone to form a straight microtubule lattice and accommodate new tubulin dimers. Conversely, microtubule ends trapped in states with unevenly long and stiff GDP-protofilaments are more prone to shortening. We conclude that protofilament clustering is a key phenomenon that links the hydrolysis state of single tubulins to the polymerization state of the entire microtubule

Choice of fluorophore affects dynamic DNA nanostructures

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modelling, we demonstrate for 12 different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation—without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices

Le Service Européen pour l’action Extérieure á l’heure de son épreuve : Une contribution tenforcée de l’UE au maintien de la paix ?

Cet article a pour objet de s’intéresser à l’impact que la mise en place d’un nouvel organe comme le Service Européen pour l’Action Extérieure, extrêmement original et innovateur du point de vue institutionnel, pourrait avoir à court et moyen terme sur la capacité de l’UE pour décoller défnitivement en tant qu’acteur majeur et partenaire vraiment crédible dans le domaine du maintien de la paix. Le moment semble opportun pour le faire dans la mesure où, après quelques quatre ans d’existence et une fois surmontés un certain nombre de teething problems, un processus de réflexion sur les faiblesses et potentialités du SEAE est en cours au sein du système institutionnel de l’UE en vue d’une éventuelle révision de sa Décision de base.En este trabajo se propone un análisis del impacto que la creación y puesta en funcionamiento de un nuevo órgano tan original e innovador desde el punto de vista institucional como el Servicio Europeo de Acción Exterior podría ejercer, a corto y medio plazo, sobre la capacidad de la UE para afanzarse como actor realmente signifcativo y socio internacional creíble en el campo del mantenimiento de la paz. El momento parece oportuno para ello en la medida en que, tras cuatro años de existencia y una vez superado un buen número de difcultades iniciales, se ha abierto un proceso de reflexión sobre las defciencias y potencialidades del SEAE que podría conducir a una revisión de su Decisión fundacional.This paper aims to analyse the impact that the setup and functioning of a new body, as original and innovative from an institutional point of view as the European External Action Service, could have in the consolidation of the EU’s position as a signifcant and reliable international partner in peacekeeping, both in the short and medium terms. This timing appears appropriate to do so insofar as, after four years of existence and having overcome a signifcant number of teething problems, a reflection process on the weaknesses and potentialities of the EEAS is currently in place and could eventually lead to a review of its founding Decision

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density

Outcomes of the EMDataResource cryo-EM Ligand Modeling Challenge

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein–nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9–2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.</p

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density