Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca 2 (HPO 4 ) 3 2 À clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories.

To date, the knowledge of the multiple roles of additives in calcium carbonate crystallization results from empirical control of crystal growth. We present a crystallization assay generating controlled supersaturation, which allows the categorization of different modes of additive action. This facilitates a novel understanding of the action of crystallization additives.

A titration set-up coupling ion selective electrodes with pH adjustment was used to analyze the effects of additives present during precipitation of calcium carbonate. Besides industrially well-established antiscalants (sodium triphosphate, citrate, polyacrylate and poly(aspartic acid)), also functional polymers being active in morphosynthesis (polystyrene sulfonate and poly(styrene-alt-maleic acid)) were analyzed. Interestingly each additive acts in its specific way, suggesting the notation "fingerprinting" for a complex interplay of up to five "solution modes" of influencing CaCO(3) precipitation and crystallisation. The results provide new insights into the modes of additive controlled crystallisation, and in the long run, the insights may facilitate the design of precipitation systems that yield complex and tailor-made crystals.

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca2+. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31-N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1-T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization.

A nacre protein, n16.3, forms phases that introduce textured mineral overgrowth and subsurface nanoporosities within calcite crystals.