Validation of Crystallographic B Factors and Analysis of Ribosomal Crystal Structures

In X-ray crystallography, validation tools assess the quality and the reliability of the structural models that crystallographers build and refine. These tools check both the consistency of physical, chemical and statistical properties of the model with the prior knowledge available in structural databases, and the agreement of the model with the diffraction data. B factors give important information about the spatial disorder of each atom around its rest position in a crystal, allowing one to infer the precision of atomic coordinates and dynamical properties of the macromolecule. The first part of the thesis work is focused on the development of a new validation tool for the distribution of isotropic B factors in crystallographic models. By means of a Bayesian approach the shifted Inverse-Gamma distribution (IGD*) is proposed as a reference distribution and a validation protocol is designed and developed to test this hypothesis. Starting from an empirical B factor distribution, the protocol returns the parameters estimates of the IGD* that best fits the B factor distribution and a p-value that is used to label the distribution as acceptable or suspicious. The protocol is then tested on a large data set of high-resolution protein structures from the PDB. From the distribution of the IGD* parameters it is possible to identify different groups of outliers, each characterized by peculiar features. Moreover, from the analysis of the distribution of p-values, the majority of the structures analysed have an acceptable B factor distribution and the agreement to the IGD* follows a hierarchical organization (whole asymmetric unit content, single chains and single domains). B factor distributions that do not satisfy the IGD* assumption usually correspond to models with problems with the deposited coordinates or diffraction data. In light of these results the developed protocol is proposed as an effective tool for the validation of B factor distributions in macromolecular crystallography. Furthermore, provided that the diffraction data are deposited in the PDB, a standard re-refinement protocol is confirmed to be a valid approach to rescue a B factor distribution from suspicious to acceptable, and to improve the quality of the results of the ensemble analysis performed with the ESCET framework if the starting data set contains models with suspicious B factor distributions. The validation protocol for B factor distributions finds a direct application in the second part of the thesis work, which is focused on the ensemble analysis with the ESCET framework of a selected data set of twenty-nine 30S ribosomal subunits from Thermus thermophilus. Thirteen refinement protocols are tested to improve, normalise and de-bias the selected structures, and to rescue models with suspicious B factor distributions. A comparative ensemble analysis is performed between the ribosomal models as deposited into the PDB and those obtained from the best refinement protocol in terms of refinement statistics and distribution of B factors. The cluster analysis is confirmed to be an effective method to automatically rationalise the structural information content of the data set. The observation that after re-refinement some structures moved to a different cluster confirms the existence of structural bias in the originally deposited structures and leads to the discovery of electron density that was not modelled in the deposited structure. Improvements of refinement statistics after re-refinement result in lower coordinate uncertainty estimates with positive effects on the results of the rigid body analysis. The main rigid bodies found on the 16S rRNA correspond to the domains known in the literature to move during the decoding process. Final remarks are given about the possible application of the presented validation tool for B factor distributions and about the importance of the availability of experimental data

Extensive counter-ion interactions seen at the surface of subtilisin in an aqueous medium

The extent of protein and counter-ion interactions in solution is still far from being fully described and understood. In low dielectric media there is documented evidence that counter-ions do bind and affect enzymatic activity. However, published crystal structures of macromolecules of biological interest in aqueous solution often do not report the presence of any counter-ions on the surface. The extent of counter-ion interactions within subtilisin in an aqueous medium has been investigated crystallographically using CsCl soak and X-ray wavelength optimised anomalous diffraction at the Cs K-edge. Ten Cs+, as well as six Cl- sites, have been clearly identified, revealing that in aqueous salt solutions ions can bind at defined points around the protein surface. The counter-ions do not generally interact with formal charges on the protein; formally neutral oxygens, mostly backbone carbonyls, mostly coordinate the Cs+ ions. The Cl- ion sites are also found likely to be near positive charges on the protein surface. The presence of counter-ions substantially changes the protein surface electrical charge. The surface charge distribution on a protein is commonly discussed in relation to enzyme function. The correct identification of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function

Molecular Rotors in a Metal–Organic Framework: Muons on a Hyper-Fast Carousel

Using muon-spin spectroscopy, we study the exceptional dynamical properties of rotating molecular struts engineered within a Zn-based metal–organic framework at cryogenic temperatures, where the internal motions of almost any other organic substance are quenched. Muon-spin spectroscopy is particularly suited for this aim, as the experimental evidence suggests several implantation sites for the muons, among which at least one directly onto the rotating moiety. The dynamics of the molecular rotors are characterized by the exceptionally low activation energy EA ∼ 30 cal mol–1. At the same time, we evidence a highly unusual temperature dependence of the dipolar interaction of muons with nuclear magnetic moments on the rotors, suggesting a complex influence of the rotations on the muon implantation and diffusion

Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals

<div><p>Azole antifungals, known as demethylase inhibitors (DMIs), target sterol 14α-demethylase (CYP51) in the ergosterol biosynthetic pathway of fungal pathogens of both plants and humans. DMIs remain the treatment of choice in crop protection against a wide range of fungal phytopathogens that have the potential to reduce crop yields and threaten food security. We used a yeast membrane protein expression system to overexpress recombinant hexahistidine-tagged <i>S</i>. <i>cerevisiae</i> lanosterol 14α-demethylase and the Y140F or Y140H mutants of this enzyme as surrogates in order characterize interactions with DMIs. The whole-cell antifungal activity (MIC₅₀ values) of both the <i>R</i>- and <i>S</i>-enantiomers of tebuconazole, prothioconazole (PTZ), prothioconazole-desthio, and oxo-prothioconazole (oxo-PTZ) as well as for fluquinconazole, prochloraz and a racemic mixture of difenoconazole were determined. <i>In vitro</i> binding studies with the affinity purified enzyme were used to show tight type II binding to the yeast enzyme for all compounds tested except PTZ and oxo-PTZ. High resolution X-ray crystal structures of ScErg11p6×His in complex with seven DMIs, including four enantiomers, reveal triazole-mediated coordination of all compounds and the specific orientation of compounds within the relatively hydrophobic binding site. Comparison with CYP51 structures from fungal pathogens including <i>Candida albicans</i>, <i>Candida glabrata</i> and <i>Aspergillus fumigatus</i> provides strong evidence for a highly conserved CYP51 structure including the drug binding site. The structures obtained using <i>S</i>. <i>cerevisiae</i> lanosterol 14α-demethylase in complex with these agrochemicals provide the basis for understanding the impact of mutations on azole susceptibility and a platform for the structure-directed design of the next-generation of DMIs.</p></div

Stereochemistry and binding of the plant pathogen CYP51 inhibitor Difenoconazole.

<p>(A) Stereoisomers of DFC, (<b>2<i>S</i>,4<i>R</i></b>) - 1-(((2<i>S</i>,4<i>R</i>)-2-(2-chloro-4-(4-chlorophenoxy)phenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1<i>H</i>-1,2,4-triazole; (<b>2<i>S</i>,4<i>S</i></b>) 1-(((2<i>S</i>,4<i>S</i>)-2-(2-chloro-4-(4-chlorophenoxy)phenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1<i>H</i>-1,2,4-triazole; (<b>2<i>R</i>,4<i>S</i></b>) 1-(((2<i>R</i>,4<i>S</i>)-2-(2-chloro-4-(4-chlorophenoxy)phenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1<i>H</i>-1,2,4-triazole; (<b>2<i>R</i>,4<i>R</i></b>) 1-(((2R,4R)-2-(2-chloro-4-(4-chlorophenoxy)phenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1H-1,2,4-triazole. (B) Orientation of DFC stereoisomers (purple carbons, PDB ID:5EAH) bound within the active site of ScErg11p6×His. The heme cofactor and selected residues (Y126, Y140 and F384) are shown as sticks. Hydrogen bonds are shown as yellow dashed lines. Helix I is shown as a grey ribbon at bottom right. (C) Rotated view showing the projection of the 4-methyl substituent towards either Y126 or the heme cofactor. Nitrogen atoms are blue, oxygen red and chlorine green.</p

MIC₅₀ data for compounds against <i>Saccharomyces cerevisiae</i> strains.

<p>MIC₅₀ data for compounds against <i>Saccharomyces cerevisiae</i> strains.</p

Chemical structures of investigated inhibitors.

<p>Chemical structures of investigated inhibitors.</p

Phytopathogen inhibitors complexed in the active site of ScERG11p6×His.

<p>The structures reveal the active site binding orientation of the enantiomers (A) <i>S</i>-TBZ (green carbons, PDB ID:5EAB), (B) <i>R</i>-TBZ (cyan carbons, PDB ID:5EAC), and (C) the superimposition of <i>S</i>-TBZ and <i>R</i>-TBZ, the active site orientation of the enantiomers (D) <i>S</i>-DPZ (magenta carbons, PDB ID:5EAD), (E) <i>R</i>-DPZ (salmon carbons, PDB ID:5EAE), and (F) the superimposition of <i>S</i>-DPZ and <i>R</i>-DPZ. The heme cofactor and selected residues (Y126 and Y140) are shown as sticks. Water-mediated (w743, red sphere) hydrogen bonds are shown as yellow dashed lines. Helix I is shown as a yellow ribbon at the bottom right of each panel. Nitrogen atoms are blue, oxygen red and chlorine green.</p

Yeast Mitochondrial Protein–Protein Interactions Reveal Diverse Complexes and Disease-Relevant Functional Relationships

Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum–mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes