Institute Biologia Molecular Of Barcelona Ibmb Csic

Barcelona, Spain

Institute Biologia Molecular Of Barcelona Ibmb Csic

Barcelona, Spain
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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.

PubMed | Institute Biologia Molecular Of Barcelona Ibmb Csic and Emil Warburg Weg 6
Type: Journal Article | Journal: Acta crystallographica. Section A, Foundations and advances | Year: 2015

Statistical tests are applied for the detection of systematic errors in data sets from least-squares refinements or other residual-based reconstruction processes. Samples of the residuals of the data are tested against the hypothesis that they belong to the same distribution. For this it is necessary that they show the same mean values and variances within the limits given by statistical fluctuations. When the samples differ significantly from each other, they are not from the same distribution within the limits set by the significance level. Therefore they cannot originate from a single Gaussian function in this case. It is shown that a significance cutoff results in exactly this case. Significance cutoffs are still frequently used in charge-density studies. The tests are applied to artificial data with and without systematic errors and to experimental data from the literature.

Propper K.,University of Gottingen | Propper K.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Meindl K.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Sammito M.,Institute Biologia Molecular Of Barcelona Ibmb Csic | And 5 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures. © 2014 International Union of Crystallography.

PubMed | Institute Biologia Molecular Of Barcelona Ibmb Csic, Autonomous University of Barcelona and European Synchrotron Radiation Facility
Type: Journal Article | Journal: 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 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.

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.

GrOtsch H.,Institute Biologia Molecular Of Barcelona Ibmb Csic | GrOtsch H.,University of Lübeck | 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.

Herms A.,In equip | Bosch M.,In equip | Ariotti N.,University of Queensland | Reddy B.J.N.,University of California at Irvine | And 15 more authors.
Current Biology | Year: 2013

Lipid droplets (LDs) are dynamic organelles that collect, store, and supply lipids [1]. LDs have a central role in the exchange of lipids occurring between the cell and the environment and provide cells with substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules such as lipoproteins or hormones. However, lipid-derived metabolites also cause progressive lipotoxicity [2], accumulation of reactive oxygen species (ROS), endoplasmic reticulum stress, mitochondrial malfunctioning, and cell death [2]. Intracellular accumulation of LDs is a hallmark of prevalent human diseases, including obesity, steatosis, diabetes, myopathies, and arteriosclerosis [3]. Indeed, nonalcoholic fatty liver disease is the most common cause of abnormal hepatic function among adults [4, 5]. Lipotoxicity gradually promotes cellular ballooning and disarray, megamitochondria, accumulation of Mallory's hyaline in hepatocytes, and inflammation, fibrosis, and cirrhosis in the liver. Here, using confocal microscopy, serial-block-face scanning electron microscopy, and flow cytometry, we show that LD accumulation is heterogeneous within a cell population and follows a positive skewed distribution. Lipid availability and fluctuations in biochemical networks controlling lipolysis, fatty acid oxidation, and protein synthesis contribute to cell-to-cell heterogeneity. Critically, this reversible variability generates a subpopulation of cells that effectively collect and store lipids. This high-lipid subpopulation accumulates more LDs and more ROS and reduces the risk of lipotoxicity to the population without impairing overall lipid homeostasis, since high-lipid cells can supply stored lipids to the other cells. In conclusion, we demonstrate fat storage compartmentalization within a cell population and propose that this is a protective social organization to reduce lipotoxicity. © 2013 Elsevier Ltd.

Rodriguez D.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Sammito M.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Meindl K.,Institute Biologia Molecular Of Barcelona Ibmb Csic | De Ilarduya I.M.,Institute Biologia Molecular Of Barcelona Ibmb Csic | And 4 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2012

Since its release in September 2009, the structure-solution program ARCIMBOLDO, based on the combination of locating small model fragments such as polyalanine -helices with density modification with the program SHELXE in a multisolution frame, has evolved to incorporate other sources of stereochemical or experimental information. Fragments that are more sophisticated than the ubiquitous main-chain - helix can be proposed by modelling side chains onto the main chain or extracted from low-homology models, as locally their structure may be similar enough to the unknown one even if the conventional molecular-replacement approach has been unsuccessful. In such cases, the program may test a set of alternative models in parallel against a specified figure of merit and proceed with the selected one(s). Experimental information can be incorporated in three ways: searching within ARCIMBOLDO for an anomalous fragment against anomalous differences or MAD data or finding model fragments when an anomalous substructure has been determined with another program such as SHELXD or is subsequently located in the anomalous Fourier map calculated from the partial fragment phases. Both sources of information may be combined in the expansion process. In all these cases the key is to control the workflow to maximize the chances of success whilst avoiding the creation of an intractable number of parallel processes. A GUI has been implemented to aid the setup of suitable strategies within the various typical scenarios. In the present work, the practical application of ARCIMBOLDO within each of these scenarios is described through the distributed test cases. © International Union of Crystallography 2012.

Giblin J.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Fernandez-Golbano I.M.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Idrissi F.-Z.,Institute Biologia Molecular Of Barcelona Ibmb Csic | Geli M.I.,Institute Biologia Molecular Of Barcelona Ibmb Csic
Biochemical Society Transactions | Year: 2011

Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-IMyo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites. ©The Authors Journal compilation ©2011 Biochemical Society.

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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