Accurate model and ensemble refinement using cryo-electron microscopy maps and Bayesian inference.

Converting cryo-electron microscopy (cryo-EM) data into high-quality structural models is a challenging problem of outstanding importance. Current refinement methods often generate unbalanced models in which physico-chemical quality is sacrificed for excellent fit to the data. Furthermore, these techniques struggle to represent the conformational heterogeneity averaged out in low-resolution regions of density maps. Here we introduce EMMIVox, a Bayesian inference approach to determine single-structure models as well as structural ensembles from cryo-EM maps. EMMIVox automatically balances experimental information with accurate physico-chemical models of the system and the surrounding environment, including waters, lipids, and ions. Explicit treatment of data correlation and noise as well as inference of accurate B-factors enable determination of structural models and ensembles with both excellent fit to the data and high stereochemical quality, thus outperforming state-of-the-art refinement techniques. EMMIVox represents a flexible approach to determine high-quality structural models that will contribute to advancing our understanding of the molecular mechanisms underlying biological functions.

Osteocalcin: Promoter or Inhibitor of Hydroxyapatite Growth?

Osteocalcin is the most abundant noncollagenous bone protein and the functions in bone remineralization as well as in inhibition of bone growth have remained unclear. In this contribution, we explain the dual role of osteocalcin in the nucleation of new calcium phosphate during bone remodeling and in the inhibition of hydroxyapatite crystal growth at the molecular scale. The mechanism was derived using pH-resolved all-atom models for the protein, phosphate species, and hydroxyapatite, along with molecular dynamics simulations and experimental and clinical observations. Osteocalcin binds to (hkl) hydroxyapatite surfaces through multiple residues, identified in this work, and the fingerprint of binding residues varies as a function of the (hkl) crystal facet and pH value. On balance, the affinity of osteocalcin to hydroxyapatite slows down crystal growth. The unique tricalcium γ-carboxylglutamic acid (Gla) domain hereby rarely adsorbs to hydroxyapatite surfaces and faces instead toward the solution. The Gla domain enables prenucleation of calcium phosphate for new bone formation at a slightly acidic pH of 5. The growth of prenucleation clusters of calcium phosphate continues upon increase in pH value from 5 to 7 and is much less favorable, or not observed, on the native osteocalcin structure at and above neutral pH values of 7. The results provide mechanistic insight into the early stages of bone remodeling from the molecular scale, help inform mutations of osteocalcin to modify binding to apatites, support drug design, and guide toward potential cures for osteoporosis and hyperosteogeny.

Determination of the Structure and Dynamics of the Fuzzy Coat of an Amyloid Fibril of IAPP Using Cryo-Electron Microscopy.

In recent years, major advances in cryo-electron microscopy (cryo-EM) have enabled the routine determination of complex biomolecular structures at atomistic resolution. An open challenge for this approach, however, concerns large systems that exhibit continuous dynamics. To address this problem, we developed the metadynamic electron microscopy metainference (MEMMI) method, which incorporates metadynamics, an enhanced conformational sampling approach, into the metainference method of integrative structural biology. MEMMI enables the simultaneous determination of the structure and dynamics of large heterogeneous systems by combining cryo-EM density maps with prior information through molecular dynamics, while at the same time modeling the different sources of error. To illustrate the method, we apply it to elucidate the dynamics of an amyloid fibril of the islet amyloid polypeptide (IAPP). The resulting conformational ensemble provides an accurate description of the structural variability of the disordered region of the amyloid fibril, known as fuzzy coat. The conformational ensemble also reveals that in nearly half of the structural core of this amyloid fibril, the side chains exhibit liquid-like dynamics despite the presence of the highly ordered network backbone of hydrogen bonds characteristic of the cross-ß structure of amyloid fibrils.

Accurate and Ultrafast Simulation of Molecular Recognition and Assembly on Metal Surfaces in Four Dimensions.

Understanding molecular interactions with metal surfaces in high reliability is critical for the development of catalysts, sensors, and therapeutics. Obtaining accurate experimental data for a wide range of surfaces remains a critical bottleneck and quantum-mechanical data remain speculative due to high uncertainties and limitations in scale. We report molecular dynamics simulations of adsorption energies and assembly of organic molecules on elemental metal surfaces using the INTERFACE force field (IFF). The force field-based simulations reach up to 8 times higher accuracy than density functional calculations at a million-fold faster speed, as well as more than 1 order of magnitude higher accuracy than other force fields relative to accurate measurements by single-crystal adsorption calorimetry. Uncertainties of prior computational methods are effectively reduced from on the order of 100% to less than 10% and validated by experimental data from multiple sources. Specifically, we describe the molecular interactions of benzene and naphthalene with even and defective platinum surfaces across a wide range of surface coverage in depth. We discuss molecular-scale influences on the heat of adsorption and clarify the definition of surface coverage. The methods can be applied to 18 metals to accurately predict binding and assembly of organic molecules, ligands, electrolytes, biological molecules, and gases without additional fit parameters.

Patterning of Self-Assembled Monolayers of Amphiphilic Multisegment Ligands on Nanoparticles and Design Parameters for Protein Interactions.

Functionalization of nanoparticles with specific ligands is helpful to control specific diagnostic and therapeutic responses such as protein adsorption, cell targeting, and circulation. Precision delivery critically depends on a fundamental understanding of the interplay between surface chemistry, ligand dynamics, and interaction with the biochemical environment. Due to limited atomic-scale insights into the structure and dynamics of nanoparticle-bound ligands from experiments, relationships of grafting density and ligand chemistry to observable properties such as hydrophilicity and protein interactions remain largely unknown. In this work, we uncover how self-assembled monolayers (SAMs) composed of multisegment ligands such as thioalkyl-PEG-(N-alkyl)amides on gold nanoparticles can mimic mixed hydrophobic and hydrophilic ligand coatings, including control of patterns, hydrophilicity, and specific recognition properties. Our results are derived from molecular dynamics simulations with the INTERFACE-CHARMM36 force field at picometer resolution and comparisons to experiments. Small changes in ligand hydrophobicity, via adjusting the length of the N-terminal alkyl groups, tune water penetration by multiples and control superficial ordering of alkyl chains from 0 to 70% regularity. Further parameters include the grafting density of the ligands, curvature of the nanoparticle surfaces, type of solvent, and overall ligand length, which were examined in detail. We explain the thermodynamic origin of the formation of heterogeneous patterns of multisegment ligand SAMs and illustrate how different degrees of ligand order on the nanoparticle surface affect interactions with bovine serum albumin. The resulting design principles can be applied to a variety of ligand chemistries to customize the behavior of functionalized nanoparticles in biological media and enhance therapeutic efficiency.

Amyloid-like amelogenin nanoribbons template mineralization via a low-energy interface of ion binding sites.

Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of ß-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the Ca­Ca distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.

Binding mechanism and binding free energy of amino acids and citrate to hydroxyapatite surfaces as a function of crystallographic facet, pH, and electrolytes.

Hydroxyapatite (HAP) is the major mineral phase in bone and teeth. The interaction of individual amino acids and citrate ions with different crystallographic HAP surfaces has remained uncertain for decades, creating a knowledge gap to rationally design interactions with peptides, proteins, and drugs. In this contribution, we quantify the binding mechanisms and binding free energies of the 20 end-capped natural amino acids and citrate ions on the basal (001) and prismatic (010)/(020) planes of hydroxyapatite at pH values of 7 and 5 for the first time at the molecular scale. We utilized over 1500 steered molecular dynamics simulations with highly accurate potentials that reproduce surface and hydration energies of (hkl) hydroxyapatite surfaces at different pH values. Charged residues demonstrate a much higher affinity to HAP than charge-neutral species due to the formation of superficial ion pairs and ease of penetration into layers of water molecules on the mineral surface. Binding free energies range from 0 to -60 kJ/mol and were determined with ∼ 10% uncertainty. The highest affinity was found for citrate, followed by Asp(-) and Glu(-), and followed after a gap by Arg(+), Lys(+), as well as by His(+) at pH 5. The (hkl)-specific area density of calcium ions, the protonation state of phosphate ions, and subsurface directional order of the ions in HAP lead to surface-specific binding patterns. Amino acids without ionic side groups exhibit weak binding, between -3 and 0 kJ/mol, due to difficulties to penetrate the first layer of water molecules on the apatite surfaces. We explain recognition processes that remained elusive in experiments, in prior simulations, discuss agreement with available data, and reconcile conflicting interpretations. The findings can serve as useful input for the design of peptides, proteins, and drug molecules for the modification of bone and teeth-related materials, as well as control of apatite mineralization.

Involvement of prenucleation clusters in calcium phosphate mineralization of collagen.

Involvement of thermodynamically-stable prenucleation clusters (PNCs) in the biomineralization of collagen has been speculated since their existence was reported in mineralization systems. It has been hypothesized that intrafibrillar mineralization proceeds via nucleation of inhibitor-stabilized intermediates produced by liquid-liquid separation (aka. polymer-induced liquid precursors; PILPs). Here, the contribution of PNCs and PILPs to calcium phosphate intrafibrillar mineralization of collagen was examined in a model with a semipermeable membrane that excludes nucleation inhibitor-stabilized PILPs from reaching the collagen fibrils, using cryogenic electron microscopy of reconstituted fibrils and conventional transmission electron microscopy of collagen sponges. Molecular dynamics simulation with the Interface force field (IFF) was used to confirm the existence of PILPs with amorphous calcium phosphate and elucidate details of the dynamics. Furthermore, intrafibrillar mineralization of single collagen fibrils was experimentally observed with unstabilized PNCs when anionic/cationic polyelectrolytes were used to establish Donnan equilibrium across the semipermeable membrane. Molecular dynamics simulation verified PNC formation within the collagen intrafibrillar gap zones at the atomic scale and explained the role of external PILPs. The PILPs decrease the interfibrillar water content and increase the interfibrillar ionic concentration. Nevertheless, intrafibrillar mineralization of collagen sponges with PNCs alone was inefficacious, being constrained by competition from extrafibrillar mineral precipitation. STATEMENT OF SIGNIFICANCE: Compared with conventional PILP-based intrafibrillar mineralization, mineralization of collagen fibrils using unstabilized PNCs is constrained by competition from extrafibrillar mineral deposition. The narrow window of opportunity for PNCs to produce intrafibrillar mineralization provides a plausible explanation for the feasibility of nucleation inhibitor-free intrafibrillar apatite assembly during reconstitution of type I collagen.

Vaporizable endoskeletal droplets via tunable interfacial melting transitions.

Liquid emulsion droplet evaporation is of importance for various sensing and imaging applications. The liquid-to-gas phase transformation is typically triggered thermally or acoustically by low-boiling point liquids, or by inclusion of solid structures that pin the vapor/liquid contact line to facilitate heterogeneous nucleation. However, these approaches lack precise tunability in vaporization behavior. Here, we describe a previously unused approach to control vaporization behavior through an endoskeleton that can melt and blend into the liquid core to either enhance or disrupt cohesive intermolecular forces. This effect is demonstrated using perfluoropentane (C5F12) droplets encapsulating a fluorocarbon (FC) or hydrocarbon (HC) endoskeleton. FC skeletons inhibit vaporization, whereas HC skeletons trigger vaporization near the rotator melting transition. Our findings highlight the importance of skeletal interfacial mixing for initiating droplet vaporization. Tuning molecular interactions between the endoskeleton and droplet phase is generalizable for achieving emulsion or other secondary phase transitions, in emulsions.