High-resolution NMR Studies Research Articles

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.

10B and 11B high-resolution NMR studies on boron-doped diamond

11B magic-angle spinning (MAS) NMR experiments are applied to B-doped diamond samples prepared by high-pressure and high-temperature methods. From the spectrum, we show that there are at least four boron signal components and the one at 28.5ppm is ascribed to the substitutional boron in the diamond structure providing the carriers responsible for conductivity. We further apply two-dimensional (2D) NMR to examine 1H dipolar broadening and 11B–11B boron spin diffusion, and candidates purported so far for the excess boron, that is, a boron+hydrogen complex and –B–B– and/or –B–C–B– clusters are negated. Furthermore, we show that 10B MAS NMR is useful to selectively observe the substitutional boron in the diamond structure appearing at 28.5ppm, whose quadrupolar coupling is much smaller than that of the excess boron at 65.5ppm.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b 5 fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a “misfolded” form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b 5 fusion protein system for the efficient production of peptides for high resolution NMR studies.

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D 2O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.

10B/ 11B 1D/2D solid-state high-resolution NMR studies on boron-doped diamond

In this work, three boron-doped diamond samples prepared from a high-pressure high-temperature synthesis method with the same starting materials but with different initial ratios for boron are studied. Magnetic susceptibility measurement shows that the increment of the initial amount of boron does not straightforwardly bring lower superconducting transition temperature. In accordance with our previous 11B high-resolution magic-angle spinning (MAS) NMR results, we show that there are at least four boron signal components and the one at 28.5 ppm is ascribed to the substitutional boron in the diamond structure providing the carriers responsible for conductivity. From observed relative intensities of the four signal components, it is suggested that the excess boron, which does not contribute to the conductivity appears as a broad signal at around 65.5 ppm. We apply two-dimensional (2D) NMR to examine 1H dipolar broadening and 11B– 11B boron spin diffusion, and candidates purported so far for the excess boron, that is, a boron + hydrogen complex and –B–B– and/or –B–C–B– clusters are negated. From its chemical-shift value, it is suggested that the excess boron exists as trigonally coordinated boron. We further apply 2D nutation NMR to examine the size of its quadrupolar coupling and show that it is not very large. As for a structure that compromises the trigonal coordination invoked from the chemical-shift value and the small quadrupolar coupling, we postulate boron locally in a graphite-like structure but the symmetry of the electric field gradient around it is high. Furthermore, we show that 10B MAS NMR is useful to selectively observe the substitutional boron in the diamond structure appearing at 28.5 ppm, whose quadrupolar coupling is much smaller than that of the excess boron at 65.5 ppm.

Structure, Activity, and Stability of Triphenyl Phosphine-Modified Rh/SBA-15 Catalyst for Hydroformylation of Propene: A High-Resolution Solid-State NMR Study

A ligand (triphenyl phosphine, PPh3)-modified heterogeneous PPh3−Rh(CO)/SBA-15 catalyst and supported Wilkinson complex HRh(CO)(PPh3)3/SBA-15 catalyst were prepared and examined in the hydroformylation of propene. Heterogeneous PPh3−Rh(CO)/SBA-15 catalyst showed much higher activity and stability in this reaction. Multinuclear 1H, 29Si, 31P, and 17O MAS NMR and two-dimensional 17O MQ MAS NMR together with XRD and N2 adsorption were employed to study the local structures of these two catalysts. Quantitative 1H and 29Si MAS NMR and qualitative one- and two-dimensional 17O MAS and MQ MAS NMR indicate that in the presence of CO the silanols on the surface of SBA-15 can react with rhodium carbonyls to form the Si−O−Rh bonds at the interface between the catalyst and the support. 31P MAS NMR spectra demonstrate a similar Wilkinson complex structure is produced on the heterogeneous PPh3−Rh(CO)/SBA-15 catalyst. The formation of Si−O−Rh bonds at the interface may immobilize the Rh complex during the long reaction. These may be correlated to the higher performances of heterogeneous PPh3−Rh(CO)/SBA-15 catalyst in propene hydroformylation.

NMR study of the cataract-linked P23T mutant of human γD-crystallin shows minor changes in hydrophobic patches that reflect its retrograde solubility

The Pro23 to Thr (P23T) mutation in human γD-crystallin (HGD) shows several cataract phenotypes. We found earlier [A. Pande, O. Annunziata, N. Asherie, O. Ogun, G.B. Benedek, J. Pande, Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin, Biochemistry 44 (2005) 2491–2500] that the mutation dramatically lowers the solubility of P23T but the overall protein fold is maintained. Recently we observed that solutions of P23T showed liquid–liquid phase transition behavior similar to that of HGD but the liquid–protein crystal phase transition was altered, suggesting an asymmetric distribution of “sticky” patches on the protein surface [J.J. McManus, A. Lomakin, O. Ogun, A. Pande, M. Basan, J. Pande, G.B. Benedek, Altered phase diagram due to a single point mutation in human gammaD-crystallin, Proc. Natl. Acad. Sci. USA 104 (2007) 16856–16861]. Here we present high-resolution NMR studies of HGD and P23T in which we have made nearly complete backbone assignments. The data provide a structural basis for explaining the retrograde solubility of P23T by (a) identifying possible “sticky” patches on the surface of P23T and (b) highlighting their asymmetric distribution.

Solid-State NMR on a Type III Antifreeze Protein in the Presence of Ice

Antifreeze proteins (AFPs) are found in fish, insects, plants, and a variety of other organisms where they serve to prevent the growth of ice at subzero temperatures. Type III AFPs cloned from polar fishes have been studied extensively with X-ray crystallography, liquid-state NMR, and site directed mutagenesis and are, therefore, among the best characterized AFPs. A flat surface on the protein has previously been proposed to be the ice-binding site of type III AFP. The detailed nature of the ice binding remains controversial since it is not clear whether only polar or also hydrophobic residues are involved in ice binding and there is no structural information available of a type III AFP bound to ice. Here we present a high-resolution solid-state NMR study of a type III AFP (HPLC-12 isoform) in the presence of ice. The chemical-shift differences we detected between the frozen and the nonfrozen state agree well with the proposed ice-binding site. Furthermore, we found that the (1)H T(1) of HPLC-12 in frozen solution is very long compared to typical (1)H of proteins in the solid state as for example of ubiquitin in frozen solution.

High resolution NMR study of T1 magnetic relaxation dispersion. I. Theoretical considerations of relaxation of scalar coupled spins at arbitrary magnetic field

A theoretical approach to the description of longitudinal (T(1)) relaxation in scalar coupled systems of spin 1/2 nuclei at arbitrary magnetic field is developed, which is based on the Redfield theory. The consideration is addressed to field-cycling relaxometry experiments with high-resolution NMR detection, in which the field dependence of T(1)-relaxation times, the nuclear magnetic relaxation dispersion (NMRD), can be studied for individual spins of the molecule. Our study reveals well-pronounced effects of spin-spin couplings on the NMRD curves. First, coupled spins having completely different high-field T(1) times tend to relax at low field with a common relaxation time. Second, the NMRD curves exhibit sharp features at the fields corresponding to the positions of nuclear spin level anticrossings. Such effects of spin-spin couplings show up not only for individual spins but also for the T(1)-relaxation of the total spin magnetization of the molecule. The influence of spin-spin coupling is of importance as long as the coupling strength J is larger than the inverse T(1)-relaxation times of the spins. Around J x T(1) = 1 there is also a coherent contribution to the relaxation kinetics resulting in an oscillatory component of the kinetic curves. Application of the theory to experimental examples will be described in subsequent publications.

Effect of Leucine to Phenylalanine Substitution on the Nonpolar Face of a Class A Amphipathic Helical Peptide on Its Interaction with Lipid: HIGH RESOLUTION SOLUTION NMR STUDIES OF 4F-DIMYRISTOYLPHOSPHATIDYLCHOLINE DISCOIDAL COMPLEX

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

The hydration features of carbohydrate determinants of Lewis antigens

The hydration behaviour of two Lewis carbohydrate determinants, Lewis X (Lex) (1a) and Lewis Y (Ley) (1b), have been investigated by a comparative 20 ns molecular dynamics (MD) study. The detailed hydration of the two oligosaccharides was described using the AMBER-based GLYCAM04 force field which is developed specifically for modelling carbohydrates. In comparison with previous in vacuo MD simulations and high resolution NMR studies, the solvated structures have shown to be dynamically stable and equally rigid with regards to glycosidic linkage torsion angles. Comparisons have also been made between the solvated structures and crystal structures currently available, namely the Lex dimer crystal, the crystal structure of Lex complex with the 291-2G3-A-Fab antibody; and the crystal structures of Ley complexed with the hu3S193 and CBR96 antibodies. When the solvated structures and respective crystal structures were overlaid for comparison the only significant difference was the Galp(3)-O5-C5-C6-O6 and the GlcpNAc(2)-O5-C5-O6-C6 orientations. The hydration sphere was investigated using a radial pair distribution function. Several solute heteroatoms were identified for the occurrence of bridging events where a solute heteroatom is linked to (at least) one other via a solvent molecule.

Dependence of the Size of a Protein−SDS Complex on Detergent and Na+ Concentrations

Sodium dodecyl sulfate (SDS) micelles provide ideal mimetic media for high-resolution NMR studies of membrane proteins and proteins or peptides interacting with micellar aggregates. (15)N NMR relaxation of the backbone amides of a protein-SDS complex has been measured under different experimental conditions. The rotational diffusion time of this complex has been found highly sensitive to detergent and NaCl concentrations. A comparison with calculated rotational diffusion times of protein-free SDS micelles under the same conditions suggests that the size of both aggregates must follow a similar functional dependence on detergent/NaCl concentration.

Degradation of copolymers obtained by ring-opening polymerization of glycolide and ɛ-caprolactone: A high resolution NMR and ESI-MS study

Electrospray ionization-mass spectrometry (ESI-MS) and proton nuclear magnetic resonance ( 1H NMR) have been used to investigate the hydrolytic degradation of copolymers obtained by bulk ring-opening copolymerization of glycolide and ɛ-caprolactone with monomer ratios ranging from 70/30 to 30/70. NMR allows changes of the average sequence distribution and composition of the components to be followed. In contrast, ESI-MS is able to reveal the detailed chemical structures of various sequences despite the molecular weight limit of 2000 Da. Combination of ESI-MS with NMR can thus provide information to describe microstructure changes during degradation. The distribution of various oligomers shown in the form of planar projections is of great interest for the design of biodegradable system aimed at medical applications.

15N and 13C High-Resolution Solid-State NMR Study of the Polymorphism of the l-Enantiomer of N-Benzoylphenylalanine

In this paper, several approaches which allow the investigation of mixtures of polymorphs, employing modern solid-state NMR (SS NMR) spectroscopy are reported. A convenient methodology for characterization of the hydrogen bonding and molecular conformation of a polymorphic sample by means of one-dimensional and two-dimensional, 13C and 15N NMR experiments as well as CSA tensor analysis and theoretical calculations is presented. Two-dimensional heteronuclear SS NMR allowed definition of the polymorphic domain of N-benzoyl-L-phenylalanine (N-Bz-Phe). The graphical method of Herzfeld and Berger was used to measure the 13C and 15N spinning sideband intensities which allowed the calculation of NMR parameters for labeled centers of N-Bz-Phe. The experimental data were compared with computed results obtained by means of the DFT hybrid method with B3PW91 functional and 6-311++G** basis set.

Structure determinations of human and chimaeric antibodies by solution scattering and constrained molecular modelling

X-ray and neutron scattering and analytical ultracentrifugation provide multiparameter structural and compositional information on proteins that complements high-resolution protein crystallography and NMR studies. They are ideal methods to use when either a large protein cannot be crystallized, when scattering provides the only means to obtain a solution structure, or the protein crystal structure has been determined and it is necessary to validate this. Once these results have been obtained, we apply automated constrained modelling methods based on known subunit crystal structures to identify the best-fit structure. Using our antibody structures as examples, we describe the generation of appropriate starting models, randomizing these for trial-and-error scattering fits, identifying the final best-fit models and interpreting these in terms of function. We discuss our structure determinations for IgA and IgD, an IgA-human serum albumin complex, the dimer of IgA and secretory component associated with this and chimaeras of mouse IgG with two complement proteins. Constrained modelling confirms the experimental data analysis and produces families of best-fit molecular models. Its usage has clarified several aspects of antibody structure and function in solution.

Surface Properties Are Highly Sensitive to Small pH Induced Changes in the 3-D Structure of α-Lactalbumin

The change in structure of bovine alpha-lactalbumin in environments of decreasing pH from pH 7 to pH 3 was followed using high-resolution NMR and hydrogen exchange studies. The effect of the changes in the structure on the surface properties of the protein was also measured. As the pH was decreased from pH 7 toward pH 2, at which alpha-lactalbumin adopts a molten globule state, a small but increasing proportion of the molecules in the sample partially unfold. There was on average a loss of tertiary structure and a change in the environment of the tryptophan residues. A significant proportion of the change measured by both circular dichroism spectroscopy and interfacial methods observed as the pH was decreased from pH 7 to pH 4 was found to be irreversible upon readjustment back to pH 7. These changes in the sample conferred an increase in surface hydrophobicity and affected the surface properties. The surface activity was found to be highest at pH 4. This was because the increasing flexibility and surface hydrophobicity of the molecule with decreasing pH was balanced by the simultaneous increase in net charge repulsion. This conclusion was also confirmed by measurements of surface shear rheology. Interestingly the interfacial dilatational rheology was highest at the isoelectric point, indicating the dominant role of the charge interaction in controlling this parameter.

Polymorphism of linezolid: A combined single-crystal, powder diffraction and NMR study

Linezolid ( S)- N-[[3-(3-fluoro-4-morpholinylphenyl)-2-oxo-5-oxazolidinyl]methyl] acetamide is one of the first commercially available (and most widely used) oxazolidinone antibiotics. It was selectively prepared as two anhydrous polymorphic forms, labelled form II and IV in accordance with preliminary reports in the patent literature. Form II has been characterized by single-crystal X-ray diffraction methods (orthorhombic, P2 12 12 1, a = 6.536(1), b = 9.949(1), c = 24.807(3) Å, V = 1613.1(3) Å 3, Z = 4, Z′ = 1), while powders of form IV could be fully characterized by employing ab initio powder diffraction methods (triclinic, P1, a = 6.5952(7) Å, b = 10.9875(10) Å, c = 12.9189(14) Å, α = 110.683(4)°, β = 88.186(6)°, γ = 105.826(6)°, V = 840.5(2) Å 3, Z = Z′ = 2). The interconversion of form II into form IV was studied by TG, DSC and thermodiffractometry, which indicated a quantitative (endothermic and irreversible) transformation (in air) just above 160 °C. On cooling from the melt, linezolid gives an oily material, stable at RT, which can be crystallized into form IV by controlled heating near 100 °C. These materials were further characterized by high-resolution 1H and 13C NMR studies, as well as by 13C solid-state NMR.

Commonality and Biosynthesis of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.

Structural Role of Fluoride in Aluminophosphate Sol−Gel Glasses: High-Resolution Double-Resonance NMR Studies

The local structure of Na-Al-P-O-F glasses, prepared by a novel sol-gel route, was extensively investigated by advanced solid-state NMR techniques. 27Al{19F} rotational echo double resonance (REDOR) results indicate that the F incorporated into aluminophosphate glass is preferentially bonded to octahedral Al units and results in a significant increase in the concentration of six-coordinated aluminum. The extent of Al-F and Al-O-P connectivities are quantified consistently by analyzing 27Al{31P} and 27Al{19F} REDOR NMR data. Two distinct types of fluorine species were identified and characterized by various 19F{27Al}, 19F{23Na}, and 19F{31P} double resonance experiments, which were able to support peak assignments to bridging (Al-F-Al, -140 ppm) and terminal (Al-F, -170 ppm) units. On the basis of the detailed quantitative dipole-dipole coupling information obtained, a comprehensive structural model for these glasses is presented, detailing the structural speciation as a function of composition.