Advanced materials engineering in historical gypsum plaster formulations.

We show how historical gypsum plaster preparation methods affect the microstructure and the wettability properties of the final stucco materials. We reproduced a traditional Persian recipe (Gach-e Koshteh, ~14th century AD), which involves a continuous mechanical treatment during plaster hydration. These samples were compared with a laboratory-replicated historical recipe from Renaissance Italy (Gesso Sottile, ~15th century AD) and contemporary low-strength plaster. The Koshteh recipe induces the formation of gypsum platelets, which exhibit preferential orientation in the plaster bulk. In contrast, the Italian and low-strength plasters comprise a typical needle-like morphology of gypsum crystals. The platelets in Koshteh expose the more hydrophilic {010} face of gypsum in a much more pronounced manner than needles. Consequently, the Iranian plaster displays enhanced wettability, enabling its direct use for water-based decoration purposes, or as a fine finishing thin layer, without the need of mixing it with a binder material. Contrary, in Sottile, gypsum crystals are left to equilibrate in large excess of water, which promotes the growth of long needles at the expense of smaller crystals. Typically, such needles are several times longer than those found in a control regular plaster. For this crystal habit, the total surface of hydrophilic faces is minimized. Consequently, such plaster layers tend to repel water, which can then be used, e.g., as a substrate for oil-based panel paintings. These findings highlight the development of advanced functional materials, by tuning their microtexture, already during the premodern era.

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

Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into two- and 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.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

Seeds of imperfection rule the mesocrystalline disorder in natural anhydrite single crystals.

In recent years, we have come to appreciate the astounding intricacies associated with the formation of minerals from ions in aqueous solutions. In this context, a number of studies have revealed that the nucleation of calcium sulfate systems occurs nonclassically, involving the aggregation and reorganization of nanosized prenucleation species. In recent work, we have shown that this particle-mediated nucleation pathway is actually imprinted in the resultant micrometer-sized CaSO4 crystals. This property of CaSO4 minerals provides us with the unique opportunity to search for evidence of nonclassical nucleation pathways in geological environments. In particular, we focused on large anhydrite crystals extracted from the Naica Mine in Mexico. We were able to shed light on this mineral's growth history by mapping defects at different length scales. Based on this, we argue that the nanoscale misalignment of the structural subunits, observed in the initial calcium sulfate crystal seeds, propagates through different length scales both in morphological, as well as in strictly crystallographic aspects, eventually causing the formation of large mesostructured single crystals of anhydrite. Hence, the nonclassical nucleation mechanism introduces a "seed of imperfection," which leads to a macroscopic "single" crystal whose fragments do not fit together at different length scales in a self-similar manner. Consequently, anisotropic voids of various sizes are formed with very well-defined walls/edges. However, at the same time, the material retains in part its single crystal nature.

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

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.

High mobility of lattice molecules and defects during the early stage of protein crystallization.

Protein crystals are expected to be useful not only for their molecular structure analysis but also as functional materials due to their unique properties. Although the generation and the propagation of defects during crystallization play critical roles in the final properties of protein crystals, the dynamics of these processes are poorly understood. By time-resolved liquid-cell transmission electron microscopy, we observed that nanosized crystal defects are surprisingly mobile during the early stages of the crystallization of a lysozyme as a model protein. This highly dynamic behavior of defects reveals that the lattice molecules are mobile throughout the crystal structure. Moreover, the disappearance of the defects indicated that intermolecular bonds can break and reform rapidly with little energetic cost, as reported in theoretical studies. All these findings are in marked contrast to the generally accepted notion that crystal lattices are rigid with very limited mobility of individual lattice molecules.

Two types of amorphous protein particles facilitate crystal nucleation.

Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

Role of clusters in nonclassical nucleation and growth of protein crystals.

The development of multistep nucleation theory has spurred on experimentalists to find intermediate metastable states that are relevant to the solidification pathway of the molecule under interest. A great deal of studies focused on characterizing the so-called "precritical clusters" that may arise in the precipitation process. However, in macromolecular systems, the role that these clusters might play in the nucleation process and in the second stage of the precipitation process, i.e., growth, remains to a great extent unknown. Therefore, using biological macromolecules as a model system, we have studied the mesoscopic intermediate, the solid end state, and the relationship that exists between them. We present experimental evidence that these clusters are liquid-like and stable with respect to the parent liquid and metastable compared with the emerging crystalline phase. The presence of these clusters in the bulk liquid is associated with a nonclassical mechanism of crystal growth and can trigger a self-purifying cascade of impurity-poisoned crystal surfaces. These observations demonstrate that there exists a nontrivial connection between the growth of the macroscopic crystalline phase and the mesoscopic intermediate which should not be ignored. On the other hand, our experimental data also show that clusters existing in protein solutions can significantly increase the nucleation rate and therefore play a relevant role in the nucleation process.

Step Crowding Effects Dampen the Stochasticity of Crystal Growth Kinetics.

Crystals grow by laying down new layers of material which can either correspond in size to the height of one unit cell (elementary steps) or multiple unit cells (macrosteps). Surprisingly, experiments have shown that macrosteps can grow under conditions of low supersaturation and high impurity density such that elementary step growth is completely arrested. We use atomistic simulations to show that this is due to two effects: the fact that the additional layers bias fluctuations in the position of the bottom layer towards growth and by a transition, as step height increases, from a 2D to a 3D nucleation mechanism.

Mesoscopic Impurities Expose a Nucleation-Limited Regime of Crystal Growth.

Nanoscale self-assembly is naturally subject to impediments at the nanoscale. The recently developed ability to follow processes at the molecular level forces us to resolve older, coarse-grained concepts in terms of their molecular mechanisms. In this Letter, we highlight one such example. We present evidence based on experimental and simulation data that one of the cornerstones of crystal growth theory, the Cabrera-Vermilyea model of step advancement in the presence of impurities, is based on incomplete physics. We demonstrate that the piercing of an impurity fence by elementary steps is not solely determined by the Gibbs-Thomson effect, as assumed by Cabrera-Vermilyea. Our data show that for conditions leading up to growth cessation, step retardation is dominated by the formation of critically sized fluctuations. The growth recovery of steps is counter to what is typically assumed, not instantaneous. Our observations on mesoscopic impurities for lysozyme expose a nucleation-dominated regime of growth that has not been hitherto considered, where the system alternates between zero and near-pure velocity. The time spent by the system in arrest is the nucleation induction time required for the step to amass a supercritical fluctuation that pierces the impurity fence.

Controlling the selective formation of calcium sulfate polymorphs at room temperature.

Calcium sulfate is a naturally abundant and technologically important mineral with a broad scope of applications. However, controlling CaSO4 polymorphism and, with it, its final material properties still represents a major challenge, and to date there is no universal method for the selective production of the different hydrated and anhydrous forms under mild conditions. Herein we report the first successful synthesis of pure anhydrite from solution at room temperature. We precipitated calcium sulfate in alcoholic media at low water contents. Moreover, by adjusting the amount of water in the syntheses, we can switch between the distinct polymorphs and fine-tune the outcome of the reaction, yielding either any desired CaSO4 phase in pure state or binary mixtures with predefined compositions. This concept provides full control over phase selection in CaSO4 mineralization and may allow for the targeted fabrication of corresponding materials for use in various areas.

Organic solvent-free synthesis of calcium sulfate hemihydrate at room temperature.

Calcium sulfate hemihydrate, also known as bassanite or Plaster of Paris, is one of the most extensively produced inorganic materials worldwide. Nowadays, bassanite is mainly obtained by thermal dehydration of calcium sulfate dihydrate (gypsum) - a process that consumes considerable amounts of energy and thus leaves a significant carbon footprint. Towards a more sustainable future, alternative technologies for bassanite production at low temperatures are therefore urgently required. While successful approaches involving organic solvents have been reported, we chose precipitation from aqueous solutions as a potentially even "greener" way of synthesis. In a previous work, we have shown that spontaneous formation of bassanite in water (in competition with thermodynamically favoured gypsum) can be achieved at 40 °C by the use of additives that maintain specific interactions with calcium sulfate precursors and modulate the local hydration household during crystallisation. The results of the present study demonstrate that bassanite can be obtained via simple precipitation from aqueous solutions at room temperature by the combination of additives acting through orthogonal mechanisms.

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

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.

Nucleation of protein mesocrystals via oriented attachment.

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

Nucleation of protein crystals - a nanoscopic perspective.

Macromolecular phase transitions bear great medical, scientific and industrial relevance, yet the molecular picture of their earliest beginnings is still far from complete. For decades, progress has been hampered by the challenges associated with studying stochastic nucleation phenomena occurring on nanoscopic length scales. In the last 5 years, however, the field has advanced with great strides due to the recent buildout of experimental techniques that allow us to observe details of the nucleation process on the nanoscale. In this review, we present a historical overview and state-of-the-art analysis of protein crystal nucleation from an experimentalist's perspective. After a short introduction of key concepts from classical nucleation theory, we discuss the advancements that have led to the development of alternative models of protein nucleation. We summarize the experimental proof in favour of these various models, but we also focus on some of their shortcomings and experimental blind spots. In our penultimate section we highlight recent works that have provided direct nanoscopic insight into the nucleation of protein crystals. We end with concluding paragraphs discussing outstanding questions and possible strategies to advance the field further in the future.

Formation of calcium sulfate through the aggregation of sub-3 nanometre primary species.

The formation pathways of gypsum remain uncertain. Here, using truly in situ and fast time-resolved small-angle X-ray scattering, we quantify the four-stage solution-based nucleation and growth of gypsum (CaSO4·2H2O), an important mineral phase on Earth and Mars. The reaction starts through the fast formation of well-defined, primary species of <3 nm in length (stage I), followed in stage II by their arrangement into domains. The variations in volume fractions and electron densities suggest that these fast forming primary species contain Ca-SO4-cores that self-assemble in stage III into large aggregates. Within the aggregates these well-defined primary species start to grow (stage IV), and fully crystalize into gypsum through a structural rearrangement. Our results allow for a quantitative understanding of how natural calcium sulfate deposits may form on Earth and how a terrestrially unstable phase-like bassanite can persist at low-water activities currently dominating the surface of Mars.

The Molecular Mechanism of Iron(III) Oxide Nucleation.

A molecular understanding of the formation of solid phases from solution would be beneficial for various scientific fields. However, nucleation pathways are still not fully understood, whereby the case of iron (oxyhydr)oxides poses a prime example. We show that in the prenucleation regime, thermodynamically stable solute species up to a few nanometers in size are observed, which meet the definition of prenucleation clusters. Nucleation then is not governed by a critical size, but rather by the dynamics of the clusters that are forming at the distinct nucleation stages, based on the chemistry of the linkages within the clusters. This resolves a longstanding debate in the field of iron oxide nucleation, and the results may generally apply to oxides forming via hydrolysis and condensation. The (molecular) understanding of the chemical basis of phase separation is paramount for, e.g., tailoring size, shape and structure of novel nanocrystalline materials.

In situ observation of biotite (001) surface dissolution at pH 1 and 9.5 by advanced optical microscopy.

Laser confocal differential interference contrast microscopy (LCM-DIM) allows for the study of the reactivity of surface minerals with slow dissolution rates (e.g., phyllosilicates). With this technique, it is possible to carry out in situ inspection of the reacting surface in a broad range of pH, ionic strength and temperature providing useful information to help unravel the dissolution mechanisms of phyllosilicates. In this work, LCM-DIM was used to study the mechanisms controlling the biotite (001) surface dissolution at pH 1 (11 and 25 °C) and pH 9.5 (50 °C). Step edges are the preferential sites of dissolution and lead to step retreat, regardless of the solution pH. At pH 1, layer swelling and peeling takes place, whereas at pH 9.5 fibrous structures (streaks) form at the step edges. Confocal Raman spectroscopy characterization of the reacted surface could not confirm if the formation of a secondary phase was responsible for the presence of these structures.

Observing classical nucleation theory at work by monitoring phase transitions with molecular precision.

It is widely accepted that many phase transitions do not follow nucleation pathways as envisaged by the classical nucleation theory. Many substances can traverse intermediate states before arriving at the stable phase. The apparent ubiquity of multi-step nucleation has made the inverse question relevant: does multistep nucleation always dominate single-step pathways? Here we provide an explicit example of the classical nucleation mechanism for a system known to exhibit the characteristics of multi-step nucleation. Molecular resolution atomic force microscopy imaging of the two-dimensional nucleation of the protein glucose isomerase demonstrates that the interior of subcritical clusters is in the same state as the crystalline bulk phase. Our data show that despite having all the characteristics typically associated with rich phase behaviour, glucose isomerase 2D crystals are formed classically. These observations illustrate the resurfacing importance of the classical nucleation theory by re-validating some of the key assumptions that have been recently questioned.