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Henn J.,Emil Warburg Weg 6 | Meindl K.,Institute Biologia Molecular Of Barcelona Ibmb Csic
Acta Crystallographica Section A: Foundations and Advances | Year: 2014

The formerly introduced theoretical R values [Henn & Schönleber (2013). Acta Cryst. A69, 549-558] are used to develop a relative indicator of systematic errors in model refinements, R meta, and applied to published charge-density data. The counter of R meta gives an absolute measure of systematic errors in percentage points. The residuals (I o - I c)/σ(I o) of published data are examined. It is found that most published models correspond to residual distributions that are not consistent with the assumption of a Gaussian distribution. The consistency with a Gaussian distribution, however, is important, as the model parameter estimates and their standard uncertainties from a least-squares procedure are valid only under this assumption. The effect of correlations introduced by the structure model is briefly discussed with the help of artificial data and discarded as a source of serious correlations in the examined example. Intensity and significance cutoffs applied in the refinement procedure are found to be mechanisms preventing residual distributions from becoming Gaussian. Model refinements against artificial data yield zero or close-to-zero values for R meta when the data are not truncated and small negative values in the case of application of a moderate cutoff I o > 0. It is well known from the literature that the application of cutoff values leads to model bias [Hirshfeld & Rabinovich (1973). Acta Cryst. A29, 510-513]. © 2014 International Union of Crystallography.

GrOtsch H.,Institute Biologia Molecular Of Barcelona Ibmb Csic | GrOtsch H.,University of Lubeck | Giblin J.P.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Idrissi F.-Z.,Institute Biologia Molecular Of Barcelona Ibmb Csic | And 8 more authors.
EMBO Journal | Year: 2010

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C ext) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C ext. The TH1 domain prevented binding of the Myo5 C ext to the yeast WIP homologue Vrp1, Myo5 C ext -induced actin polymerization and recruitment of the Myo5 C ext to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C ext. Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization. © 2010 European Molecular Biology Organization.

Encinar del Dedo J.,University of Salamanca | Idrissi F.-Z.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Arnaiz-Pita Y.,University of Salamanca | James M.,SUNY Upstate Medical University | And 6 more authors.
Traffic | Year: 2014

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module. © 2014 John Wiley & Sons A/S.

Martinelli L.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Martinelli L.,Netherlands Cancer Institute | Garcia-Morales L.,Autonomous University of Barcelona | Querol E.,Autonomous University of Barcelona | And 4 more authors.
PLoS Pathogens | Year: 2016

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle. © 2016 Martinelli et al.

Henn J.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Meindl K.,Institute Biologia Molecular Of Barcelona Ibmb Csic
Acta Crystallographica Section A: Foundations and Advances | Year: 2014

In order to detect and graphically visualize the absence or presence of systematic errors in fit data, conditional probabilities are employed to analyze the statistical independence or dependence of fit residuals. This concept is completely general and applicable to all scientific fields in which model parameters are fitted to experimental data. The applications presented in this work refer to published charge-density data. © 2014 International Union of Crystallography.

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