1,721,003 research outputs found
Quantifying radiation damage in X-ray diffraction experiments in structural biology
Get PDFQuantitative studies of global radiation damage are presented for two different types of experiments in structural biology: macromolecular crystallography (MX) and small angle X-ray scattering (SAXS)
MX is the most common technique to elucidate the atomic resolution structures of biological macromolecules.
However, these molecules undergo radiation induced changes during the experiment that undesirably affect the data.
Global radiation damage, which is characterised by an overall loss in the diffracted intensity of Bragg reflections, limits the amount of useful data that can be collected from a single crystal in an experiment.
Furthermore, for experimental phasing experiments, the radiation induced intensity changes can be so significant that the phasing signal becomes undetectable, thereby hindering successful structure determination.
This thesis investigates methods to track and correct the diffraction data that are affected as a result of global radiation damage.
First, extensions to the diffraction weighted dose (DWD) metric are investigated for the ability of DWD to track the overall intensity decay of reflections.
This metric then is combined with a new mathematical model of intensity decay to perform zero-dose extrapolation.
An additional probabilistic extrapolation approach is incorporated into the traditional regression based approach to allow extrapolation of low multiplicity reflections.
As an alternative approach, a new hidden Markov model representation of the data collection experiment is developed that allows the time-resolved calculation of structure factor amplitudes, with error estimates calculated explicitly.
This method gives comparable refinement statistics to that obtained from data processed with the current data reduction pipeline, and improvements to the algorithm are proposed.
SAXS, on the other hand, is a complementary structural technique that results in low resolution information about macromolecules.
However it still requires the probing of the macromolecules with ionising radiation, so radiation induced changes are still a problem.
Unfortunately the tools for assessing radiation damage in SAXS experiments are not mature enough for them to be used routinely.
This thesis presents extensions to RADDOSE-3D to perform dose calculations for SAXS samples.
Additionally, a free, open source Python library has been developed to allow the exploration and visualisation of the results of a similarity analysis of frames within a dataset.
These tools are then used to determine the efficacy of various radioprotectant compounds at different concentrations to mitigate radiation damage effects.</p
Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy
Get PDFReactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target. Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA. The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine. By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay
Radiation damage in protein crystallography: susceptibility study
Get PDFProtein structure models obtained from X-ray crystallography are subject to radiation damage. The resulting specific alterations to protein structures can be mistaken for biological features, or may obscure actual protein mechanisms, leading to misidentification or obscuration of biological insight. The radiation chemistry behind this site-specific damage is not well understood. Radiation damage processes progress in proportion to the dose absorbed by the crystal in the diffraction experiment. Doses can be estimated using existing software, but these assume idealised experimental conditions. To simulate complex diffraction experiments, including treatment of imperfect X-ray beam profiles and inhomogeneous dose distributions, a new program, RADDOSE-3D, was developed. RADDOSE-3D can be integrated into beamline software to provide convenient, more accurate, comparative, and publishable dose figures, also facilitating informed data collection decisions. There is currently no method to automatically detect specific radiation damage in protein structure models in the absence of an 'undamaged' reference model. Radiation damage research therefore generally relies on detailed observation of a few model proteins. A new metric, BDamage, is designed and used to identify and quantify specific radiation damage in the first large-scale statistical survey of 2,704 published protein models, which are examined for the effects of local environments on site-specific radiation damage susceptibility. A significant positive correlation between susceptibility and solvent accessibility is identified. Current understanding of radiation damage progression is mostly based on a few consecutive structure model 'snapshots' at coarse dose intervals. The low sampling rate considerably limits the ability to identify varying site susceptibility and its causes. Real space electron density data are obtained for crystals of different mutants of a RhoGDI protein with very high sequence identity, to determine sensitising and stabilising factors for radiation induced structural changes. Utilising a newly developed data collection and analysis protocol, these changes could be tracked with unprecedented time resolution
The solution and crystal structures of a module pair from the Staphylococcus aureus-binding site of human fibronectin - A tale with a twist
No full textAn important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type I modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV radiation damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins. (c) 2007 Elsevier Ltd. All rights reserved
A complex iron-calcium cofactor catalyzing phosphotransfer chemistry
Get PDFAlkaline phosphatases play a crucial role in phosphate acquisition by microorganisms.To expand our understanding of catalysis by this class of enzymes, we have determined thestructure of the widely occurring microbial alkaline phosphatase PhoX. The enzymecontains a complex active-site cofactor comprising two antiferromagnetically coupledferric iron ions (Fe3+), three calcium ions (Ca2+), and an oxo group bridging three of themetal ions. Notably, the main part of the cofactor resembles synthetic oxide-centeredtriangular metal complexes. Structures of PhoX-ligand complexes reveal how theactive-site metal ions bind substrate and implicate the cofactor oxo group in thecatalytic mechanism. The presence of iron in PhoX raises the possibility that ironbioavailability limits microbial phosphate acquisition.Fil: Yong, Shee Chien. University of Oxford; Reino UnidoFil: Roversi, Pietro. University of Oxford; Reino UnidoFil: Lillington, James E.D.. University of Oxford; Reino UnidoFil: Rodriguez, Fernanda Mariana. University of Oxford; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Krehenbrink, Martin. University of Oxford; Reino UnidoFil: Zeldin, Oliver B.. University of Oxford; Reino UnidoFil: Garman, Elspeth F.. University of Oxford; Reino UnidoFil: Lea, Susan M.. University of Oxford; Reino UnidoFil: Berks, Ben C.. University of Oxford; Reino Unid
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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