Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors

M.S. acknowledges financial support from the Fonds Wetenschappelijk Onderzoek (FWO) under Project Nos. G0H5316N and 1516215N. This work used the platforms of the Grenoble Instruct-European Research Infrastructure Consortium (ERIC) center (Integrated Structural Biology Grenoble [ISBG]; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INBS-0005-02) and Grenoble Alliance for Integrated Structural and Cell Biology (GRAL), financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) Chemistry, Biology, and Health, European Graduate School (CBHEUR-GS) (ANR-17-EURE-0003). The electron microscope facility is supported by the Auvergne-Rhone-Alpes Region, the Fondation pour la Recherche Medicale (FRM), the Fonds Europeen de Developpement Regional (FEDER), and the Groupement d'Interet Scientifique (GIS)-Infrastructures en Biologie Sante et Agronomie. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG), Le Commissariata l'Energie Atomique et aux Energies Alternatives (CEA).Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into twoand three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed.FWO G0H5316N 1516215NFrench Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005-02Grenoble Alliance for Integrated Structural and Cell Biology (GRAL)within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) Chemistry, Biology, and Health, European Graduate School (CBHEUR-GS) ANR-17-EURE-000

Evidence for liquid-liquid phase separation during the early stages of Mg-struvite formation

We thank BAM and Helmholtz-Zentrum Berlin (HZB) for providing us with the beamtime at mySpot of BESSY II.The supplementary material document file contains the following items: Fig. S1. The time-resolved pH curve from the struvite precipitation reaction [Eq. (1)] combined with PHREEQC calculated equilibrated pH; Fig. S2. Cryo-TEM bright-field imaging from the reactant solution sampled after 5 s after mixing without any post-processing. Fig. S3. Sketch of the flow-through setup used for the scattering experiments; Supporting Note 1: We derive how the OZ correlation length compares with an equivalent radius of a sphere. We also include input (MgStruvite_01_input.pqi) and output (MgStruvite_output.pqo) files from PHREEQC 3, which contain information about the predicted speciation at equilibrium following the reaction from Eq. (1). These are regular text files and are human-readable.The precipitation of struvite, a magnesium ammonium phosphate hexahydrate (MgNH4PO4 · 6H2O) mineral, from wastewater is a promising method for recovering phosphorous. While this process is commonly used in engineered environments, our understanding of the underlying mechanisms responsible for the formation of struvite crystals remains limited. Specifically, indirect evidence suggests the involvement of an amorphous precursor and the occurrence of multi-step processes in struvite formation, which would indicate non-classical paths of nucleation and crystallization. In this study, we use synchrotron-based in situ x-ray scattering complemented by cryogenic transmission electron microscopy to obtain new insights from the earliest stages of struvite formation. The holistic scattering data captured the structure of an entire assembly in a time-resolved manner. The structural features comprise the aqueous medium, the growing struvite crystals, and any potential heterogeneities or complex entities. By analysing the scattering data, we found that the onset of crystallization causes a perturbation in the structure of the surrounding aqueous medium. This perturbation is characterized by the occurrence and evolution of Ornstein-Zernike fluctuations on a scale of about 1 nm, suggesting a non-classical nature of the system. We interpret this phenomenon as a liquid-liquid phase separation, which gives rise to the formation of the amorphous precursor phase preceding actual crystal growth of struvite. Our microscopy results confirm that the formation of Mg-struvite includes a short-lived amorphous phase, lasting >10 s.Helmholtz-Zentrum für Umweltforschung UF

Determining the operational window of green antiscalants: A case study for calcium sulfate

The detrimental effects of inorganic scaling in industrial and domestic applications are often mitigated with scale inhibitors. Increasing environmental awareness and stringent regulations require developing more sustainable antiscalants. Testing of suitable candidates is often the rate-limiting step in development cycles, therefore we developed a high-throughput methodology to rapidly evaluate the antiscaling potential of new additives under different application conditions. Using this method we determined the performance of two potential green additives – a chelating agent and a threshold inhibitor – in delaying CaSO4 precipitation over a wide range of supersaturations, temperatures and salinities. The threshold inhibitor strongly delayed CaSO4 scaling, but its performance is highly dependent on the physicochemical conditions, with the appropriate application window comprising low salinities and mild temperatures. In contrast, the chelating agent showed a lower inhibiting capacity, but its performance remained relatively constant throughout the entire matrix of physicochemical conditions. Noteworthy, we also observed that at intermediate salinities the absolute induction time for CaSO4 precipitation is dramatically prolonged, offering a sustainable strategy to mitigate scaling. Overall, our method allows simultaneously benchmarking the scaling kinetics and testing the scale-inhibiting performance of additives, providing a direct route to a more rational design of antiscaling technologies.BI-OTIC campus de excelencia (UGR) 30.BA.94.080

Hierarchical synchrotron diffraction and imaging study of the calcium sulfate hemihydrate–gypsum transformation

ISTerre is part of Labex OSUG@2020. Use of the Geochem- istry–Mineralogy platform at ISTerre is acknowledged. The authors wish to aknowledge the ESRF for provision of beam time (MA4498). We are also thankful to Dr Pierre-Olivier Autran for useful insights on tomographic reconstructions and Dr Marta Majkut for discussions on grain orientation calcu- lations. The authors also thank Dr Irina Snigireva and Dr Nathaniel Findling for SEM characterization of the samples and Dr Catherine Dejoie for complementary high-resolution powder diffraction data from the ID22 beamline of the ESRF.The mechanism of hydration of calcium sulfate hemihydrate (CaSO4 0.5H2O) to form gypsum (CaSO4 2H2O) was studied by combining scanning 3D X-ray diffraction (s3DXRD) and phase contrast tomography (PCT) to determine in situ the spatial and crystallographic relationship between these two phases. From s3DXRD measurements, the crystallographic structure, orientation and position of the crystalline grains in the sample during the hydration reaction were obtained, while the PCT reconstructions allowed visualization of the 3D shapes of the crystals during the reaction. This multi-scale study unfolds structural and morphological evidence of the dissolution–precipitation process of the gypsum plaster system, providing insights into the reactivity of specific crystallographic facets of the hemihydrate. In this work, epitaxial growth of gypsum crystals on the hemihydrate grains was not observed

Cluster-mediated stop-and-go crystallization

Impurities control the formation of bio-crystals and can fully paralyze crystal growth at low levels of supersaturation. Traditional impurity models predict that an escape from this so-called “dead zone” requires an increase in the driving force (i.e. supersaturation). In this work, using protein crystals as a model system, we uncover an alternative escape route from the dead zone that does not involve an increase in supersaturation. We demonstrate that the merger of a protein cluster with the crystal surface triggers the formation of an ordered multi-layered island. The newly created surface on top of the resulting 3D island is initially devoid of impurities and therefore characterized by near-pure step growth kinetics. The accelerated step advancement on this relatively uncontaminated surface limits the available time for impurities to adsorb on the emerging terraces and by extension their resulting surface density. Cluster-mediated crystal growth occurring in heterogeneous media can therefore lead to stop-and-go dynamics, which offers a new model to explain crystallization taking place under biological control (e.g. biomineralization).The European Space Agency under Contract No. ESA AO-2004-070, FWO grant 1523115 N (Belgium)Scholarship BES⋅2003⋅2191 (AVD, Ministerio de Ciencia y Tecnologia, Spain)

Experimental crystallization of trioctahedral smectites under hydrothermal conditions. Implication for alkaline hydrothermal vents

Present day hydrothermal vents are considered as analogues of sites where live could have emerged on primitive earth. Strýtan (Iceland) is a shallow hydrothermal system composed of vertical Si-Mg structures, whose mineral association corresponds to a low crystallinity silica, trioctahedral smectites (Sm) and smaller amounts of carbonates. To better understand mineral formation in such vent systems we have synthesized trioctahedral Sm in the kerolite (Krl)-stevensite (Stv)-saponite (Sap) series using hydrothermal conditions in slightly alkaline solutions from amorphous hydrous Mg-Al silicates. The starting material consisted of coprecipitated gels with different Si/Mg/Al/Na atomic ratios. Gels were aged with water for 60 days at 150, 175, 200 and 225 °C in Teflon lined reactors. The solid samples were characterized by XRD, DTA-TG, FTIR and TEM. XRD revealed the formation of low crystallinity phyllosilicates. The 001 reflection evolved with increasing T and Al from 9.7, to 12 and 14.5 Å. The patterns contained peaks corresponding to Krl and Stv/Sap. In oriented mounts, solvation with ethylene glycol shifted the 001 peak to 17.4 Å in gels 2 and 3, whereas in Gel 1 it remained at 9.7 Å with a shoulder at 17.2 Å. FTIR bands were also associated to talc-Krl and Stv-Sap. TEM images of Gel 1 at 225°C showed unresolved aggregates of tiny crystals of 10 nm, with turbostratic structures. Gel 3 produced layered crystals containing numerous defects and with spacings of 10-11 Å, typical of trioctahedral Sm. Thus, aging treatment transformed gels into Krl-Sm interstratified minerals and trioctahedral Sm. T increased the conversion rate from 40 to 75% at 150°C to 225°, respectively. Gel 1 produced a 20-80% Stv-Krl interstratified mineral, composed of tiny crystals. Increasing Al content improved the crystallinity and the % of the Sm. Gel 2 consisted of a Sm-Krl interstratified mineral richer in Sm, while Gel 3 is fully transformed into Sap with tetrahedral Al

Mechanisms and kinetics of C-S-H nucleation approaching the spinodal line: Insights into the role of organics additives

Wet chemistry C-S-H precipitation experiments were performed under controlled conditions of solution supersaturation in the presence and absence of gluconate and three hexitol molecules. Characterization of the precipitates with SAXS and cryo-TEM experiments confirmed the presence of a multi-step nucleation pathway. Induction times for the formation of the amorphous C-S-H spheroids were determined from light transmittance. Analysis of those data with the classical nucleation theory revealed a significant increase of the kinetic prefactor in the same order as the complexation constants of calcium and silicate with each of the organics. Finally, two distinct precipitation regimes of the C-S-H amorphous precursor were identified: i) a nucleation regime at low saturation indexes (SI) and ii) a spinodal nucleation regime at high SI where the free energy barrier to the phase transition is found to be of the order of the kinetic energy or less.Comment: Accepted in Cement and Concrete Research. 30 pages plus supplementary materials. arXiv admin note: substantial text overlap with arXiv:2111.0274

On the Quality of Protein Crystals Grown under Diffusion Mass-transport Controlled Regime (I)

It has been previously shown that the diffraction quality of protein crystals strongly depends on mass transport during their growth. In fact, several studies support the idea that the higher the contribution of the diffusion during mass transport, the better the diffraction quality of the crystals. In this work, we have compared the crystal quality of two model (thaumatin and insulin) and two target (HBII and HBII-III) proteins grown by two different methods to reduce/eliminate convective mass transport: crystal growth in agarose gels and crystal growth in solution under microgravity. In both cases, we used identical counterdiffusion crystallization setups and the same data collection protocols. Additionally, critical parameters such as reactor geometry, stock batches of proteins and other chemicals, temperature, and duration of the experiments were carefully monitored. The diffraction datasets have been analyzed using a principal component analysis (PCA) to determine possible trends in quality indicators. The relevant indicators show that, for the purpose of structural crystallography, there are no obvious differences between crystals grown under reduced convective flow in space and convection-free conditions in agarose gel, indicating that the key factor contributing to crystal quality is the reduced convection environment and not how this reduced convection is achieved. This means that the possible detrimental effect on crystal quality due to the incorporation of gel fibers into the protein crystals is insignificant compared to the positive impact of an optimal convection-free environment provided by gels. Moreover, our results confirm that the counterdiffusion technique optimizes protein crystal quality and validates both environments in order to deliver high quality protein crystals, although other considerations, such as protein/gel interactions, must be considered when defining the optimal crystallization setup.This study was supported by projects ESP2005-23831-E and ESP2007-29071-E (Spanish Ministry of Education and Science) and BIO2016-74875-P (JAG) (MINECO), Spain co-funded by the Fondo Europeo de Desarrollo Regional, FEDER funds, European Union

Effect of Solution Stoichiometry on BaSO4 Crystallization from Turbidity Measurements and Modeling

The impact of solution stoichiometry on the nucleation and growth of BaSO4 was studied by measuring solution transmittance and subsequent fitting to a crystallization model. Our results show that a large excess of either Ba2+ or SO4 2− ions inhibits both the nucleation and growth of BaSO4. However, for a small excess of Ba2+, the growth is enhanced. The dependence of nucleation and growth rates on supersaturation and solution stoichiometry was captured by a semiempirical rate model. Hence, the solution stoichiometry is a highly relevant parameter while studying all aspects of BaSO4 crystallization, and it could be worthwhile to examine other minerals similarly.European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 819588)Junta de Andalucía (PROYEXCEL_00771

Modelización computacional de la reacción de protólisis de la superficie de filosilicatos

En la interfase mineral/solución de los filosilicatos se producen reacciones de adsorción, disolución y re-precipitación, dando lugar a un cambio de la estructura del sólido y de la solución. Este estudio tiene como objetivo ampliar nuestro conocimiento sobre los mecanismos de reacción de hidrólisis sobre la superficie de modelos tipo clúster de los filosilicatos usando los cálculos de DFT. Los resultados derivados de los cálculos de la hidrólisis sobre la superficie (010) de dos estructuras, una celda de pirofilita y otra de palygorskita. Estos modelos se basaron en un clúster formado por 2 cavidades tetraédricas con un anillo de SiO4 unidas a una capa octaédrica, la diferencia fundamental entre ellas es la orientación de las unidades tetraédricas y la continuidad de la capa octaédrica. Los cálculos se realizaron utilizando Gaussian 16. Los puntos críticos de energía potencial superficial de la reacción de hidrólisis se calcularon utilizando el funcional B3LYP, aplicando la base lanl2dz. Los resultados obtenidos muestran que la hidrólisis no se produce, se observa una reacción de protólisis sobre la superficie (010). En los cálculos realizados, tomando la pirofilita, el catión H3O+ ataca a uno de los enlaces de Al-O-Al, obteniéndose dos estructuras protonadas estables. Posteriormente, se realizó un segundo ataque en otro de los enlaces puente que produce la ruptura total del modelo. No se encontró ningún estado de transición (TS) que llevara a estas estructuras estables protonadas obtenidas. Para la modelización del clúster de la palygorskita, se procedió de la misma manera y se consiguieron resultados similares, pero en este caso sí se llegó a observar un TS. Estos cálculos apoyan la hipótesis de que la superficie (010) parece ser más reactiva que la (001) de los filosilicatos 2:1 que se deriva de trabajos previos de este grupo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech