Calcium sulfate minerals such as gypsum play important roles in natural and industrial processes, but their precipitation mechanisms remain largely unexplored. We used time-resolved sample quenching and high-resolution microscopy to demonstrate that gypsum forms via a three-stage process: (i) homogeneous precipitation of nanocrystalline hemihydrate bassanite below its predicted solubility, (ii) self-assembly of bassanite into elongated aggregates co-oriented along their c axis, and (iii) transformation into dihydrate gypsum. These findings indicate that a stable nanocrystalline precursor phase can form below its bulk solubility and that in the CaSO(4) system, the self-assembly of nanoparticles plays a crucial role. Understanding why bassanite forms prior to gypsum can lead to more efficient anti-scaling strategies for water desalination and may help to explain the persistence of CaSO(4) phases in regions of low water activity on Mars.

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 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 liquidlike 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.prenucleation clusters | phase transition | self-purification T he process of crystallization is generally considered to occur in two consecutive but very different stages: nucleation and growth. The first stage was already studied more than two centuries ago by Gibbs, who considered the nucleation of water droplets from a supersaturated vapor through the formation of globulae. He was the first to develop a thermodynamic formalism of nucleation by considering the generation of nuclei of a liquid phase as a density fluctuation of the parent phase (1, 2). Since then, Gibbs' nucleation theory has been extended to the nucleation of solid phases from solution and gaseous phases from which the current paradigm (based on the capillary approximation) for nucleation emerged, i.e., the classical nucleation theory ]. There is, however, an increasing body of evidence that shows that CNT can fail drastically when used in cases where the implicit and explicit assumptions of CNT are poorly justified (for a full dissection of the limitations, see refs. 9-11). An obvious situation where CNT will have limited applicability is in cases where the old and the new phases differ by at least two order parameters, e.g., density and structure (12).Recent theoretical, computational, and experimental efforts have demonstrated that densification and local increase in crystallinity need not occur simultaneously (13)(14)(15)(16)(17)(18)(19)(20). These results have inspired the development of a new approach that considers nucleation from solution as a multistep process attributing key roles to metastabl...

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.

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 timeresolved 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.nucleation | protein | lysozyme | transmission electron microscopy | in situ observation C rystallization can be divided into two processes: nucleation and crystal growth. The crystal growth process has been well examined for a long time, yet the nucleation process is not understood; for example, the nucleation rate of crystals provides a textbook example of order-of-magnitude discrepancies between theoretical predictions and experimental results. Recent proposals have attributed these discrepancies to a nonclassical nucleation pathway, along which a structured crystalline embryo forms within a highly concentrated disordered precursor (1). This mechanism was first proposed for protein crystals (2, 3). Direct observations have demonstrated its applicability to organic (4), inorganic (5, 6), and colloidal (7) crystals. In proteins, clusters of protein molecules have been suggested as precursors; these clusters have mesoscopic sizes from several tens to several hundreds of nanometers and are considered to behave like liquids. It has also been suggested that the precursor is thermodynamically stable with respect to the mother liquid phase but is metastable or unstable with respect to the crystalline phase. The latter nature of the precursor differs from the stable macroscopically dense liquid formed as a result of the liquid-liquid phase separation (8). Such protein-rich mesoscopic clusters have been observed for many proteins, primarily using optical techniques, and have been tentatively identified as precursors for crystal nucleation (9-12). Several important questions concerning this mechanism remain unanswered. First, are the observed mesoscopic clusters actually liquid-like or solid-amorphous? Second, do they play an active role in crystal nucleation? In addition, finally, do the clusters serve as classical heteroge...

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.

We observed two-dimensional (2D) nucleation behavior on {110} and {101} faces of tetragonal crystals of model protein lysozyme by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). We measured, for the first time directly and noninvasively, the 2D nucleation rates using 99.99% pure lysozyme, 98.5% pure lysozyme (Seikagaku Co.), and 99.99% pure lysozyme with intentionally added impure proteins (fluorescent-labeled lysozyme, covalently bonded dimer of lysozyme, and 18 kDa polypeptide). We found that 2D nucleation was the dominant growth mechanism under conditions adopted in this study, and the 2D nucleation occurred randomly on the entire crystal surface irrespective of supersaturation within the range of σ ) ln(C/C e ) ) 0-1.4, where C is a bulk lysozyme concentration and C e the solubility (crystal size: 0.2-0.3 mm). Repeated 2D nucleation, which continued for 3-4 layers, was also observed mainly when the impure proteins were present. In addition, multilayered 2D islands were formed after the adsorption of relatively large foreign particles on the crystal surface. From the comparison between the 2D nucleation rates determined on the {110} faces with and without the impure proteins, we concluded that homogeneous 2D nucleation occurred under a higher supersaturation range (σ > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (σ < 0.8), we found that significant heterogeneous 2D nucleation dominated the growth mainly when the impure proteins were present. The {101} faces exhibited more significant heterogeneous 2D nucleation induced by smaller amounts of impurities than in the case of the {110} faces. We also determined the ledge free energies of the homogeneous and heterogeneous nucleation. Within the experimental conditions used in this study, we could not find significant dependence of the ledge free energies of the heterogeneous nucleation on the kinds of impure proteins.

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