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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Application of sulfur SAD to small crystals with a large asymmetric unit and anomalous substructure

The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al. (2019), Structure , 27 , 1137–1141]. Here, alternative approaches to determining the 40-atom sulfur substructure of the 103 kDa Ric-8A dimer that composes the asymmetric unit are explored. At the data-collection wavelength of 1.77 Å measured at the Frontier micro-focusing Macromolecular Crystallography (FMX) beamline at National Synchrotron Light Source II, the sulfur anomalous signal strength, |Δ ano |/σΔ ano ( d ′′/sig), approaches 1.4 at 3.4 Å resolution. The highly redundant, 11 000 000-reflection data set measured from 18 crystals was segmented into isomorphous clusters using BLEND in the CCP 4 program suite. Data sets within clusters or sets of clusters were scaled and merged using AIMLESS from CCP 4 or, alternatively, the phenix.scale_and_merge tool from the Phenix suite. The latter proved to be the more effective in extracting anomalous signals. The HySS tool in Phenix , SHELXC / D and PRASA as implemented in the CRANK 2 program suite were each employed to determine the sulfur substructure. All of these approaches were effective, although HySS , as a component of the phenix.autosol tool, required data from all crystals to find the positions of the sulfur atoms. Critical contributors in this case study to successful phase determination by SAD included (i) the high-flux FMX beamline, featuring helical-mode data collection and a helium-filled beam path, (ii) as recognized by many authors, a very highly redundant, multiple-crystal data set and (iii) the inclusion within that data set of data from crystals that were scanned over large ω ranges, yielding highly isomorphous and highly redundant intensity measurements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structures of two novel crystal forms of Aspergillus oryzae alpha amylase (taka-amylase)

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (β/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Euler characteristic as a basis for teaching topology concepts to crystallographers

The simple Euler polyhedral formula, expressed as an alternating count of the bounding faces, edges and vertices of any polyhedron, V – E + F = 2, is a fundamental concept in several branches of mathematics. Obviously, it is important in geometry, but it is also well known in topology, where a similar telescoping sum is known as the Euler characteristic χ of any finite space. The value of χ can also be computed for the unit polyhedra (such as the unit cell, the asymmetric unit or Dirichlet domain) which build, in a symmetric fashion, the infinite crystal lattices in all space groups. In this application χ has a modified form (χ m ) and value because the addends have to be weighted according to their symmetry. Although derived in geometry (in fact in crystallography), χ m has an elegant topological interpretation through the concept of orbifolds. Alternatively, χ m can be illustrated using the theorems of Harriot and Descartes, which predate the discovery made by Euler. Those historical theorems, which focus on angular defects of polyhedra, are beautifully expressed in the formula of de Gua de Malves. In a still more general interpretation, the theorem of Gauss–Bonnet links the Euler characteristic with the general curvature of any closed space. This article presents an overview of these interesting aspects of mathematics with Euler's formula as the leitmotif. Finally, a game is designed, allowing readers to absorb the concept of the Euler characteristic in an entertaining way.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2020

In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Crystal structure of a trigonal polymorph of aquadioxidobis(pentane-2,4-dionato-κ 2 O , O ′)uranium(VI)

The title compound, [UO 2 (acac) 2 (H 2 O)] consists of a uranyl(VI) unit ([O=U=O] 2+ ) coordinated to two monoanionic acetylacetonate (acac, C 5 H 7 O 2 ) ligands and one water molecule. The asymmetric unit includes a one-half of a uranium atom, one oxido ion, one-half of a water molecule and one acac ligand. The coordination about the uranium atom is distorted pentagonal–bipyramidal. The acac ligands and O w atom comprise the equatorial plane, while the uranyl O atoms occupy the axial positions. Intermolecular hydrogen bonding between complexes results in the formation of two-dimensional hexagonal void channels along the c- axis direction with a diameter of 6.7 Å. The monoclinic ( P 2 1 / c space group) polymorph was reported by Alcock & Flanders [(1987). Acta Cryst. C 43 , 1480–1483].

Hernandez, Alejandro↗
DOE OSTI2024

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here, in this work, we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel “dimer of dimers” mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Optical vibrational spectra and proposed crystal structure of ε-UO 3

ε-UO 3 is an exotic polymorph in the uranium trioxide system with an undetermined crystal structure and limited optical vibrational spectroscopic data. To improve understanding of this compound, we synthesize and investigate the crystal structure and optical vibrational spectra of ε-UO 3 . Infrared spectra collected for ε-UO 3 are in good agreement with previously published results, and our studies extend the available data into the low-energy (600–100 cm –1 ) regime. For the first time, Raman spectra are presented for ε-UO 3 using both 785 and 532 nm excitation wavelengths. Previous reports suggest an impurity phase may be present in ε-UO 3 produced by calcination of U 3 O 8 ; however, spectral center-of-mass calculations, principal component analyses, and Raman spectroscopic mapping employed to investigate this possibility indicate that the product of U 3 O 8 calcined in O 3 (g) in this work is likely phase-pure. A possible novel structure solution for ε-UO 3 is determined via Rietveld refinement of powder X-ray diffraction data and is triclinic, P-1, with a = 4.01 Å, b = 3.85 Å, c = 4.18 Å, and α = 98.26°, β = 90.41°, γ = 120.46° (R wp = 8.30%). The asymmetric unit of ε-UO 3 consists of U(VI) in hexagonal bipyramidal coordination with displaced equatorial oxygen. Further analysis reveals that the structure of ε-UO3 is best described by a 2 × 1 × 2 supercell structure in P-1 with a = 8.03 Å, b = 3.86 Å, c = 8.37 Å with α = 98.26°, β = 90.41°, and γ = 120.46°, although a higher-symmetry structure is possible. Optical vibrational spectroscopic and structural measurements of ε-UO 3 presented here furthers our understanding of this complex uranium oxide and clarifies the origin of reported structural similarity to U 3 O 8 .

-UO3↗
DOE OSTI2025

A Novel Solid Form of Erlotinib: Synthesis by Heterogeneous Complexation and Characterization by NMR Crystallography

We describe the synthesis of a novel complex of the anticancer "active pharmaceutical ingredient erlotinib (ERL) via heterogeneous nucleation on polished zinc tiles. The resulting product, ERL 2 ·ZnCl 2 , is characterized by single-crystal X-ray diffraction, multinuclear solid-state NMR (ssNMR) spectroscopy, and density functional theory (DFT) calculations. Also characterized are the hydrochloride salt (ERL·HCl) and monohydrate free base (ERL·H 2 O) forms of erlotinib. 13 C ssNMR spectroscopy is useful for site-by-site assignment and rapid fingerprinting, while also providing preliminary structural interpretations, such as the number of molecules in the asymmetric unit. 35 Cl ssNMR can readily differentiate between the chloride ions in ERL·HCl and the covalently bonded chlorine in ERL 2 ·ZnCl 2 . 15 N ssNMR proves to be critical here because of the large isotropic chemical shift differences between ERL·H 2 O, ERL·HCl, and ERL 2 ·ZnCl 2 . The 15 N chemical shift tensors are linked directly to differences in structure and bonding with the aid of DFT calculations. Together, these results demonstrate the utility of multinuclear NMR crystallography for the characterization of solid forms of APIs, especially when other analytical techniques face significant challenges.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Supramolecular Interactions of Teixobactin Analogues in the Crystal State

This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me- D -Gln 4 ,Lys 10 -teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel β-sheet dimers in the crystal lattice, with residues 1–3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Magneto-structural studies of an unusual [Mn III Mn II Gd III (OR) 4 ] 4– partial cubane from 2,2'-bis- p- t Bu-calix[4]arene

Reaction of 2,2'-bis- p - t Bu-calix[4]arene (H 8 L) with MnCl 2 ·4H 2 O, GdCl 3 ·6H 2 O and 2,6-pyridinedimethanol (H 2 pdm) affords [Mn III Mn II Gd III (H 3 L)(pdmH)(pdm)(MeOH) 2 (dmf)]·3MeCN·dmf ( 3 ·3MeCN·dmf) upon vapour diffusion of MeCN into the basic dmf/MeOH mother liquor. 3 crystallises in the tetragonal space group P 4 1 2 1 2 with the asymmetric unit comprising the entire cluster. The highly unusual core contains a triangular arrangement of Mn III Mn II Gd III ions housed within a [Mn III Mn II Gd III (OR) 4 ] 4– partial cubane. Magnetic susceptibility and magnetisation data reveal best fit parameters J Mn(II)–Mn(III) = +0.415 cm –1 , J Mn(III)–Gd(III) = +0.221 cm –1 , J Mn(II)–Gd(III) = –0.258 cm –1 and D Mn(III) = –4.139 cm –1 . Theoretically derived magnetic exchange interactions, anisotropy parameters, and magneto-structural correlations for 3 are in excellent agreement with the experimental data.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Applying Crystallography and 19 F NMR to investigate dynamics and partner protein interactions in a long chain Flavodoxin

Flavodoxin (Fld) is a small FMN containing protein that is involved in single electron transfer. The long‐chain flavodoxin in Rhodopseudomonas palustris bacteria replaces ferredoxin as a low‐potential electron carrier when iron is scarce. Thus it is proposed to interact with the bifurcating electron transfer flavoprotein (ETF) that yields low‐potential electrons. A surface loop on Fld interacts with another of Fld's partner proteins, so we hypothesize that it also mediates Fld's interaction with ETF. To monitor interactions with ETF directly and investigate dynamics in this loop, we are using 19 F NMR in solution. 19 F is hyperresponsive to changes in its chemical environment with a chemical shift range of >300 ppm. To provide a static reference point and assess structural heterogeneity, we are also exploiting X‐ray crystallography. In this study we selectively fluorinated the five tyrosine residues in Fld. We obtained resonance assignments from 19 F spectra of Fld variants in which individual tyrosine residues have been replaced. We obtain well resolved signals for each residue, but the resonances' linewidths indicate dynamics that affects some resonances more than others. Y90 residue displays two resonances demonstrating two different conformations that interconvert slowly on an NMR time scale. Meanwhile the crystal structure solved at 2.1Å resolution reveals two molecules per asymmetric unit providing two perspectives on the details of the structure. The crystal symmetry is monoclinic in contrast to most of the other Flds, which are orthorhombic. Interestingly, the long loop bearing Y121 and Y123 is not well resolved in chain B of the crystal structure, and the NMR line of Y123 is exceptionally broad, both indicating that the loop is dynamic and capable of altering its conformation to accomodate binding to a partner protein. Future directions include monitoring the changes in the 19 F NMR of the Fld when titrated with the partner protein, temperature dependence of the NMR spectrum and relaxation studies to evaluate time scales of motions.

Khan, Sharique↗
DOE OSTI2020

Crystal structure of bis(1-mesityl-1 H -imidazole-κ N 3 )diphenylboron trifluoromethanesulfonate

The solid-state structure of bis(1-mesityl-1 H -imidazole-κ N 3 )diphenylboron trifluoromethanesulfonate, C 36 H 38 BN 4 + ·CF 3 SO 3 − or ( Ph 2 B(MesIm) 2 OTf ), is reported. Bis(1-mesityl-1 H -imidazole-κ N 3 )diphenylboron ( Ph 2 B(MesIm) 2 + ) is a bulky ligand that crystallizes in the orthorhombic space group Pbcn . The asymmetric unit contains one Ph 2 B(MesIm) 2 + cationic ligand and one trifluoromethanesulfonate anion that balances the positive charge of the ligand. The tetrahedral geometry around the boron center is distorted as a result of the steric bulk of the phenyl groups. Weak interactions, such as π–π stacking are present in the crystal structure.

Kouton, Aniffa↗
DOE OSTI2021

Detection of translational noncrystallographic symmetry in Patterson functions

Detection of translational noncrystallographic symmetry (TNCS) can be critical for success in crystallographic phasing, particularly when molecular-replacement models are poor or anomalous phasing information is weak. If the correct TNCS is detected then expected intensity factors for each reflection can be refined, so that the maximum-likelihood functions underlying molecular replacement and single-wavelength anomalous dispersion use appropriate structure-factor normalization and variance terms. Here, an analysis of a curated database of protein structures from the Protein Data Bank to investigate how TNCS manifests in the Patterson function is described. These studies informed an algorithm for the detection of TNCS, which includes a method for detecting the number of vectors involved in any commensurate modulation (the TNCS order). The algorithm generates a ranked list of possible TNCS associations in the asymmetric unit for exploration during structure solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space groupP2 1 2 1 2 enabled the successful modeling of 96% of residues in the asymmetric unit, with finalR work andR free values of 0.199 and 0.229, respectively.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP 8 . IP 8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP 8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg 2+ or Mn 2+ ; IP 6 , 5-IP 7 , or 1,5-IP 8 ) and the other which was ligand-free. The ligand-free enzyme adopts an “open” conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP 6 or 5-IP 7 engagement above the nucleotide favors adoption of a “closed” conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP 7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N 6 . Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N 6 -benzyl-ATP as phosphate donor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of the E. coli agmatinase, SPEB

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E . coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P 3 1 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/R free values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Alphavirus Particles Can Assemble with an Alternate Triangulation Number

Alphaviruses are spherical, enveloped RNA viruses primarily transmitted by mosquitoes, and cause significant arthritogenic and neurotropic disease in humans and livestock. Previous reports have shown that—in contrast to prototypical icosahedral viruses—alphaviruses incorporate frequent defects, and these may serve important functions in the viral life cycle. We confirm the genus-wide pleomorphism in live viral particles and extend our understanding of alphavirus assembly through the discovery of an alternate architecture of Eastern equine encephalitis virus (EEEV) particles. The alternate T = 3 icosahedral architecture differs in triangulation number from the classic T = 4 icosahedral organization that typifies alphaviruses, but the alternate architecture maintains the quasi-equivalence relationship of asymmetric units. The fusion spike glycoproteins are more loosely apposed in the T = 3 form with corresponding changes in the underlying capsid protein lattice. This alternate architecture could potentially be exploited in engineering alphavirus-based particles for delivery of alphaviral or other RNA.

59 BASIC BIOLOGICAL SCIENCES↗