The remarkable mechanical properties of bone are determined by the organization and strength of binding at the mineral–collagen interface.

In the gap region, however, the collagen molecules are oriented approximately parallel to the unit cell c-axis (slightly tilted in the opposite direction to that of the overlap tilt).

microfibrils, which each comprise five 1D-staggered, twisted collagen triple helix molecules.

examined the structure of the collagen gap zone by analyzing the 3D electron density map created from existing X-ray data of hydrated type-I collagen fibrils32.

derive from their use of thicker samples (250 and 500 nm, respectively) and of a less sensitive camera system

This was confirmed by analysis of a model system starting from different image qualities (see Supplementary Figs. 6 and 7 for details).

Needles (~2 nm in width and ~10 nm in length, Supplementary Figs. 4 and 5) were observed at the tip of some platelets, which confirms previous observations2,12 and supports the hypothesis that the HAp platelets evolve from needle-like crystals.

include novel field emission guns, electron optics, a Volta phase plate, and an electron camera

as high–frame rate movie stacks, which allows for correction of beam-induced specimen motion and image blurring (Li et al. 2013).

As compared with charge-coupled devices, direct electron detectors do not require the conversion of electrons to photon signals

With the use of cryo-EM, there is reduced diffusion of charges and molecular fragments, leading to cryo-protection from beam damage

directly observe the structure and identify the chemical composition of biological specimens and materials with nanoscale resolution

dissolved in the phosphate solution at three different concentrations (20, 50 or 100 mg/L) before being mixed with an equal volume of the calcium counterion solution.

the effect of MW of pAsp (MW: 10.3 kDa or 32.2 kDa) on intrafibrillar collagen mineralization was reported by Jee et al.28. Using 32.2 kDa pAsp yielded faster and more mature mineralization than using 10.3 kDa pAsp, which was attributed to a higher stability of pAsp-ACP precursors produced with higher-MW pAsp.

Nucleation and growth of significant amount of extrafibrillar minerals on the collagen fibrils was obtained using these solutions.

Conversely, unstable solutions were obtained using high PAA MW at low concentrations.

Over stable PAA-ACP solutions that fully inhibited mineralization of the collagen matrices were achieved using PAA 2k-50.

Densified type I collagen films

13C DNP MAS NMR experiments reveal that dense liquid microdroplets are enriched with bicarbonate ions compared to the rest of the solution

Moreover, the strong affinity of aspartate for calcium carbonate was also shown through the use of l- and d-Asp to grow left- and right-handed vaterite toroids, respectively. (79)

For carbonates, the main correlation for CO32– and HCO3– concerns water molecules found around 6.0 ppm, higher than water from glycerol.

and glycerol (62.4 and 71.8 ppm) (Figure 10C)

We do not observe any excess of bicarbonates for these prenucleation species contrary to dense liquid microdroplets (i.e., in the absence of l-Asp).

the CO32–/HCO3– proportion does not match with the expected speciation at pH 8.7 (i.e., the pH measured after 3 min of reaction; Figure SI 12A) where bicarbonates should account for ∼20% of the signal at 100 K according to the reference experiment (Figure SI 10A).

by the low temperature and the partial deuteration as the “apparent” pKa (HCO3–/CO32–) in the DNP conditions is decreased from 10.3 to ∼6 (Figure SI 10B,C).

as shown recently for glycine precipitation. (68,69)

the increase in NMR sensitivity provided by DNP (67)

asymptotic growth rate of δ43Ca

a single 43Ca resonance is observed indicating fast chemical exchange of free and l-Asp-bonded Ca2+ at room temperature (Figure 9B

working in excess of Ca with a Ca/l-Asp molar ratio = 54),

coffee-ring effect where the evaporation of water molecules occurs from the interior to the exterior of the droplet transporting ACC nanoparticles.

tend to be deposited on the external part of the former nanodroplets

size similar to that previously reported. (60) However, this is the first time that the evolution of the CaCO3 droplets are observed in situ,

observed by Wolf et al. in levitated droplets. (58)

was first postulated by Faatz at al. (63)

at the expense of the smaller ones (through Ostwald ripening for instance).

does not seem to be due to the displacement of the drops

small with a size ranging from 7 to 15 nm

leading to the formation of a dense and extended inorganic network

display an average size of 3.9 ± 0.8 nm and tend to aggregates in 20–50 nm objects.

occurs beyond a binodal limit to form liquid nanodroplets

more favorable than direct crystallization as the excess free energy of the solid–liquid interface is lower than the solid-crystal interface. (58)

reaching a certain critical limit, the dynamics of the PNCs is greatly reduced, engendering the phase separation. (35,36,61)

happen directly from free ions in solution (59,60)

happen directly from free ions in solution

observed experimentally, and in situ even the size observed here is 1 order of magnitude larger.

the translational and rotational degrees of freedom gained by the water molecules provoke an increase in entropy, rendering the ion association an entropy driven process and not an enthalpy driven reaction.

during the multiple binding of PNCs, coordinated water is released from the hydration shells of the free Ca2+ and CO32– ions

there is no phase boundary

are linked by ionic bonds allowing the molecular precursors chains to behave like a polymer and adopt different dynamic configurations such as ring, linear, or branched structures.

Previous simulation studies (51)

Their presence is further confirmed by intensity profile analysis (Figure SI 1) and comparison with blank experiment (pure water) for which the contrast is ∼4 times weaker (i.e., signal-to-noise-ratio ∼4 times lower).

Movies were recorded 10 frames per second, and the electron dose was calculated as per the model of Schneider et al. (54)

100 mM calcium chloride solution and (2) a 10 mM

heating sodium bicarbonate (labeled 13C, 99%, Cortecnet) at 573 K under an inert atmosphere for 2

hyperpolarized solid state nuclear magnetic resonance (DNP NMR)

l-aspartic acid favors the formation of individual and aggregated prenucleation clusters