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Gavira J.A.,Laboratorio Of Estudios Cristalograficos
Archives of Biochemistry and Biophysics | Year: 2015

Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fields trying to shed some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization from a regular-laboratory point of view. It is also devoted to highlight the latest developments and achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput advances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to gather structural information from single particles such as Cryo-EM are also discussed. Finally, current approaches showing the convergence of different structural biology techniques and the cross-methodologies adaptation to tackle the most difficult problems, are presented. Synopsis: Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies applicable at laboratory scale. © 2015 Elsevier Inc.

Sleutel M.,Vrije Universiteit Brussel | Van Driessche A.E.S.,Laboratorio Of Estudios Cristalograficos
Crystal Growth and Design | Year: 2013

We report on the failure of the Cabrera-Vermilyea (CV) step pinning model to reproduce the elementary step kinetics for the case of tetragonal lysozyme crystals growing from contaminated solutions. We measured the supersaturation dependency of the step velocity using confocal microscopy for three different commercially available lysozyme batches with varying levels of impurity content, that is, Seikagaku, Fluka, and Sigma. Strong nonlinear dependencies are obtained in the high to intermediate supersaturation range and near-linear dependencies at lower driving forces. The clear absence of a dead zone for the Fluka and Seikagaku data is in direct contradiction to the CV model. As such, we developed a time-dependent impurity model based on Bliznakov kinetics assuming Langmuir adsorption. Admissible fits are obtained for Fluka and Seikagaku lysozyme corroborating the self-purification interpretation due to the diminishing terrace exposure times at higher supersaturation levels. The steeper recovery toward pure kinetics for Sigma lysozyme than predicted by Langmuir adsorption prompted us to expand the model to allow for impurity-impurity interaction. The resultant kinetic model, which assumes a Kisliuk-like mode of impurity adsorption, did yield acceptable fits with Sigma step kinetics. This Bliznakov-Kisliuk model also predicts clustering of impurity molecules on the surface, which is corroborated by our in situ experimental atomic force microscopy observations. © 2013 American Chemical Society.

Glaab F.,University of Regensburg | Kellermeier M.,University of Konstanz | Kunz W.,University of Regensburg | Morallon E.,University of Alicante | Garcia-Ruiz J.M.,Laboratorio Of Estudios Cristalograficos
Angewandte Chemie - International Edition | Year: 2012

Silica gardens are well-known examples for the self-assembly of inorganic material (see figure). The growth of hollow tubes results in the spontaneous formation of two compartments with highly dissimilar pH and ion concentrations, which cause electrochemical potential differences across the membrane. Initially generated gradients are relieved over time through dynamic diffusion and precipitation processes. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kellermeier M.,University of Konstanz | Kellermeier M.,University of Regensburg | Glaab F.,University of Regensburg | Melero-Garcia E.,Laboratorio Of Estudios Cristalograficos | Garcia-Ruiz J.M.,Laboratorio Of Estudios Cristalograficos
Methods in Enzymology | Year: 2013

Silica biomorphs and silica gardens are canonical examples of precipitation phenomena yielding self-assembled nanocrystalline composite materials with outstanding properties in terms of morphology and texture. Both types of structures form spontaneously in alkaline environments and rely on simple, and essentially similar, chemistry. However, the underlying growth processes are very sensitive to a range of experimental parameters, distinct preparation procedures, and external conditions. In this chapter, we report detailed protocols for the synthesis of these extraordinary biomimetic materials and identify critical aspects as well as advantages and disadvantages of different approaches. Furthermore, modifications of established standard procedures are reviewed and discussed with respect to their benefit for the control over morphogenesis and the reproducibility of the experiments in both cases. Finally, we describe currently used techniques for the characterization of these fascinating structures and devise promising ways to analyze their growth behavior and formation mechanisms in situ and as a function of time. © 2013 Elsevier Inc.

Kellermeier M.,University of Konstanz | Gebauer D.,University of Konstanz | Melero-Garcia E.,Laboratorio Of Estudios Cristalograficos | Drechsler M.,University of Bayreuth | And 5 more authors.
Advanced Functional Materials | Year: 2012

Calcium carbonate precipitation proceeds via a complex multistage scenario involving neutral ion clusters as precursors and amorphous phases as intermediates, which finally transform to crystals. Although the existence of stable clusters in solution prior to nucleation has been demonstrated, the molecular mechanisms by which they precipitate are still obscure. Here, direct insight into the processes that drive the transformation of individual clusters into amorphous nanoparticles is provided by progressive colloidal stabilization of different transient states in silica-containing environments. Nucleation of calcium carbonate in the presence of silica can only take place via cluster aggregation at low pH values. At higher pH, prenucleation clusters become colloidally stabilized and cannot aggregate. Nucleation through structural reorganization within the clusters is not observed under these conditions, indicating that this pathway is blocked by kinetic and/or thermodynamic means. The degree of stabilization against nucleation is found to be sufficient to allow for a dramatic enrichment of solutions with prenucleation clusters and enable their isolation into the dry state. This approach renders direct analyses of the clusters by conventional techniques possible and is thus likely to facilitate deeper insight into the chemistry and structure of these elusive species in the future. Under suitable conditions, added silica binds to ion clusters that exist in CaCO 3 solutions prior to nucleation. The resulting colloidal interactions can be tuned to either fully prevent nucleation and isolate the clusters or allow for their gradual transformation into amorphous nanoparticles. The processes underlying homogeneous nucleation of CaCO 3 become decelerated and can be observed experimentally. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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